【作者】 王玉龙；

【导师】 华育平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2009， 博士

【摘要】 禽流感是由A型流感病毒引起的禽类传染病,其易感动物包括各种家禽、宠物鸟和野生水禽。禽流感不仅给养禽业、野生鸟类种群带来严重的破坏,而且对公共卫生产生巨大威胁。野生鸟类一直被学术界认为是禽流感病毒的天然宿主,并在野鸟种群中能够持续循环存在,那么就有必要继续对野鸟种群中的禽流感病毒的流行情况、分布模式以及野鸟在禽流感病毒传播过程中的作用进行深入的调查研究与分析。本文主要从三个方面开展研究:第一部分,对黑龙江野鸟禽流感病毒分离株的HA、NA基因进行遗传进化分析。本研究的毒株分别来自2006哈尔滨市林业局送检的濒死猛禽黄爪隼和在黑龙江省三江国家自然保护区以网捕的方式重点对野生水禽和部分陆生鸟类捕获后采集咽喉棉拭子和泄殖腔棉拭子,样本经国家禽流感参考实验室鉴定,得到禽流感病毒四株,分别命名为A/Lesser Kestrel/Harbin/194/2007(H5N1)、A/Garganey/SanJiang/160/2006(H5N2)、A/Mallard/SanJiang/98/2006(H3N8)、A/Mallard/SanJiang/167/2006.(H3N8)。应用MEGA4(MEGA software)对所测各HA、NA基因序列结果与Genbank上已发表的野生鸟类中分离的H5N1、H5N2、H3N8亚型AIV和一些代表株序列进行比较分析,并构建NJ系统进化树,探讨四株不同来源的野鸟禽流感病毒株的系统进化关系。序列分析结果表明,A/Lesser Kestrel/Harbin/194/2007(H5N1)分离株HA的裂解位点插入多个碱性氨基酸,具有典型高致病性禽流感毒株基因序列的特征:A/Lesser Kestrel/Harbin/194/2007(H5N1)分离株的HA起源于家禽源H5N1病毒;NA与2005年后中国东南沿海及东南亚国家H5N1分离株同处一组。A/Garganey/SJ/160/06(HSN2)分离株HA的裂解位点的氨基酸序列符合低致病性禽流感毒株的分子特征;A/Garganey/SJ/160/06的HA基因处在第一分支,与该组中的江西和日本分离株亲缘关系较近,与欧美分离株较远。A/Garganey/SJ/160/06(H5N2)分离株NA基因遗传进化树树形与HA基因树形相似,但有所不同。A/Garganey/SJ/160/06处在第一分支,与该组中的江西分离株和部分欧美分离株亲缘关系较近。A/Mallard/SanJiang/98/2006(H3N8)和A/Mallard/SanJiang/167/2006(H3N8)分离株HA的裂解位点处的氨基酸序列符合低致病性禽流感毒株的分子特征。两株绿头鸭H3N8流感病毒分离株的HA基因属于欧亚亚系,并形成一个小的独立分支,且两株HA基因间核苷酸同源率很高。A/Mallard/SanJiang/167/2006(H3N8)流感分离株的NA基因位于第二分支,属于北美亚系,此基因可能来源于野生水禽。A/Mallard/SanJiang/98/2006(H3N8)流感分离株的NA基因位于第三分支,属于欧亚亚系,起源于家禽源。第二部分,禽流感病毒在野鸟种群中的分布模式分析。分析结果显示,目前至少已从17目130种野鸟体内分离出禽流感病毒或禽流感血清学检测呈阳性。这说明低致病性禽流感病毒已在全球范围内的野鸟种群中持续循环存在,但野生鸟类作为高致病性禽流感病毒的天然宿主的作用尚不很清楚。低致病性禽流感病毒可以在家禽和野鸟中循环变异,低致病性可转变成高致病性,直到高致病性禽流感疫情发生。此外,有证据表明野生鸟类可感染HPAI H5N1病毒,而且候鸟和留鸟都可感染。本文在总结分析以往有关禽流感病毒研究文献的基础上,旨在对全球各亚型禽流感病毒在野生鸟类中的流行情况、禽流感病毒在野鸟中的分布和循环模式、候鸟迁徙与流感病毒传播、野鸟禽流感病毒感染的季节性特征和自然环境介质在流感病毒传播中的作用等方面有更多和更深入的认识。第三部分,研究分析野鸟在高致病性H5NI亚型禽流感病毒长距离传播中的作用。本文在对野鸟隐性感染H5N1、野鸟HPAI事件、洲际间不同亚型禽流感病毒分子流行病学比较研究、以及近来新兴的交叉学科—生态免疫学的相关研究是否支持野鸟在迁徙行为中可实现对H5N1亚型禽流感病毒的长距离传播等四个方面的事件报道和相关研究数据,进行总结和分析,认为已有相关数据,不能充分支持野鸟特别是迁徙野鸟可对H5N1呈隐性感染状态。也很难支持野鸟在感染H5N1亚型禽流感病毒后可通过长距离迁徙行为传播病毒,但其可能具有在感染后小范围内短距离传播病毒的能力。同时,洲际间流感病毒基因间十分有限的交换几率,也可帮助进一步说明野鸟很难在感染H5N1亚型禽流感病毒后实现对病毒的长距离传播。而从生态免疫学的研究角度看,迁徙行为发生时,野鸟特有的生理和免疫状态,也不支持野鸟在感染高致病力禽流感病毒后仍可以完成长距离迁徙行为并实现对病毒的传播。分析结论是野鸟在感染H5N1亚型禽流感病毒后到出现死亡前可能进行短距离空间移动,以接力模式传播病毒,并实现将病毒传播给在移动途中遇到的同种或异种野鸟、家禽甚至人类。更多还原

【Abstract】 Avian influenza(AI),caused by the influenza virus type"A",can affect domestic poultry,pet birds and wild birds.It is severely danger to public health and wild bird populations.Wild birds are approved as natural hosts of AIV.And avian influenza viruses are cycling in wild birds.It’s necessary to investigate and analysize the prevalence,distribution pattern of AIV and the role of migrant birds for transmission of AIV between wild and captive and between different species.The thesis includes three parts:The first part:Surface gene phylogenic analysis of AIV isolated from wild birds inhabit in Heilongjiang areaThe samples were collected from agonal lesser kestrel submitted by Harbin forestry bureau 2006,and others from wild birds in "Sanjiang" national natural reserve in Heilongjiang province 2006.Cloacal swabs were taken from wild birds.The samples were found to be positive for influenza virus.Culturing and subsequent subtyping of viruses from these samples revealed three different haemagglutinin(HA)/neuraminidase(NA) subtypes(H5N1,H5N2,H3NS) by National Influenza Reference Laboratory.The N-J phylogenetic tree structured and analyzed using software-MEGA4,showed that HA gene of A/Lesser Kestrel/Harbin/194/2007 harbored a series of basic amino acids(-RRRKKR-) at HA cleavage site similar to the sequences characteristic of HPAIV,phylogenic analysis suggested that the HA gene of A/Lesser Kestrel/Harbin/194/2007 was originated from poultry of H5N1 in southern China.The NA gene have evolved to a separate clade of which many viruses from Southeast coast of China and southeast Asia after 2005.HA cleavage site of A/Garganey/SJ/160/06 was similar to the sequences characteristic of LPAIV,the HA gene have evolved to a first clade of which 2 viruses from Jiangxi and Japan formed a closer clade,a farther clade with those from Europe and America.The NA phylogenic tree was similar to the HA,but A/Garganey/SJ/160/06 is in clade 1,of which the viruses from Jiangxi and part of Europe and America formed a closer clade, a farther clade with those from Japan.The HA of H3N8 cleavage sites of 2 viruses from Sanjiang were similar to the sequences characteristic of LPAIV.The HA genes of H3N8 from Sanjiang were grouped together and highly similarity,belong to Eurasia lineage.The NA gene of A/Mallard/SanJiang/167/2006(H3N8) has evolved to second clade belong to North American lineage,of which Aquatic bird/Korea/JN-1/2005 formed a closer clade.The NA gene of Mallard/SanJiang/167/2006 was likely originated from aguatic bird.The NA gene of A/Mallard/SanJiang/98/2006 was in the clade 3,belongs to Eurasia lineage and was originate from poultry.The second part:Analysis of distribution pattern of AIV in wild birdsThe analysis showed that avian influenza viruses were isolated or the samples were found to be positive for influenza virus using serologic identification from at least 130 species of global wild birds.This demonstrates that wild birds play a role in the perpetuation of LPAIV;but the role of wild birds as the hosts of HPAIV is not very clear.LP virus might variate HP during cycling between domestic poultry and wild bird.In addition,not only migrant but also resident birds can infect HP subtype H5N1 virus.The paper discussed some questions base on the research data of AIV,including the prevalence of each subtype of AIV in global wild birds,distributing pattern of AW in wild birds,influenza viruses in ducks,the migratory birds and AIV transmission,seasonal characteristic of AIV infect wild birds and the role of natural environment reservoirs in the transmission of influenza viruses;so as to understand much about AIV in wild birds.The third part:Analysis the role of wild birds for long-distance transmission of HPAIV H5N1To identify whether the wild birds can play an role in highly pathogenic avian influenza(HPAI) virus H5N1 spread across different countries or even different states,the following four related research progresses included wild birds asymptomatic infected H5N1,HPAI outbreaks in wild birds, the exchanges of AW genes between north America and Eurasia,and the ecologic immunology study of migratory birds were reviewed in this paper.Based on the analysis of the reviews above,we find that:Firstly,the related studies data is few that can be used to prove wild birds can infect H5N1 but don’t show any symptoms,and no evidence from wild bird HPAI outbreaks can support they had spreaded the H5N1 over long distance on seasonal migration routes.But the migratory birds can be infected and may be involved in local spread of the highly pathogenic H5N1 virus is possible. Secondly,while influenza viruses do exchange between the two hemispheres,but it is a rare occurrence,this also can help to prove that the possibility of wild birds spread H5N1 along with theirs migratory routs for a long distance at least from Eurasia to North America is so limit.Thirdly, the obtained study results from ecological immunity of wild birds,mainly the more understanding about the special condition on physical and immune system of wild birds when migratory,cannot support they can still spread the H5N1 for long distance effectively after they infected by HPAI H5N1 virus.In conclution,wild birds could spread the highly pathogenic H5N1 virus to domestic avian,other wild birds even human beings by local movement(or relay race mode).更多还原