我国部分禽流感病毒H5N1之HA序列变异演化分析

从GenBank上获得我国人(Homo sapiens)、家禽和野鸟42株H5N1亚型禽流感病毒的HA基因核酸序列,利用DNAStar分析HA蛋白关键位点氨基酸残基的变化,比较HA基因核苷酸序列同源性,构建遗传进化树.探讨我国部分人、家禽和野鸟H5N1病毒基因的遗传进化关系.序列分析结果表明:禽流感病毒H5N1亚型的HA基因持续地发生着变异,但并非以均一速度进行,时间间隔愈长,核苷酸同源性愈低;我国同一地区或临近地区,当年或前后两年发生的人及家禽感染的禽流感病毒高度同源.推测我国部分人发生的禽流感可能是通过家禽感染的;候鸟的迁徙在传播病毒过程中所起的作用有待深入探讨.     

华东地区家鸭中N1亚型禽流感病毒NA基因的遗传进化分析

为了解华东地区家鸭内禽流感病毒的遗传进化情况,对2002~2006年分离自华东地区家鸭的3种主要N1亚型的禽流感病毒:2株H1N1、10株H3N1和14株H5N1,共26株病毒的NA基因进行了遗传进化分析。结果表明,华东地区家鸭中的N1亚型的禽流感病毒正处于不断进化状态中。14株H5N1禽流感病毒均在NA的茎部缺失20个氨基酸(49~68位),而其他N1亚型的禽流感病毒的NA都未见发生此缺失。H3N1病毒可能与H1N1病毒发生了NA基因的重排,但是目前还没有直接证据表明华东地区家鸭中H5N1禽流感参与了基因重排。     

番鸭源H6N6亚型禽流感病毒全基因组的分子特征

【目的】为了丰富水禽源禽流感病毒的分子流行病学资料,明确我国国内首次分离的番鸭源H6N6亚型禽流感(Avian influenza virus,AIV)病毒A/Muscovy Duck/Fujian/FZ01/2008(H6N6)(以下简称MD/FJ/F1/08)全基因组的分子特征,弄清该病毒的遗传进化特点。【方法】对其8个基因片段分别进行扩增和序列测定,并利用分子生物学软件对测序结果进行序列分析。【结果】MD/FJ/F1/08的HA裂解位点附近的氨基酸序列为PSMKVIV↓GL,为非连续的碱性氨基酸,其静脉接种指数(the intravenoys pathogenicity index,IVPI)为0.15,推测其为一株低致病力AIV。其HA基因、NP基因、M基因和PB2基因均与我国台湾分离株A/duck/Kingmen/E322/04(H6N2)该基因的核苷酸同源性最高,分别高达94.2%、95.7%、97.2%和95.6%,均处于同一遗传进化分支。其NA基因和我国远东分离株A/duck/Eastern China/01/2007(H4N6)同源性最高,达97.1%;其颈部有11个氨基酸的缺失(TNSTTTIINNN),为N6亚型神经氨酸酶基因中首次报道,在遗传进化上和H4N6亚型AIV的NA基因处于相同的分支。NS基因和香港地区分离株A/duck/HongKong/3600/99(H6N2)同源性最高,达96.1%;PB1和PA均与高致病性禽流感病毒株A/duck/HongKong/140/1998(H5N1)同源性最高,达95.6%和96.7%。且MD/FJ/F1/08的8基因与H6N6亚型流感病毒北美洲分离代表株均不处在同一遗传进化分支上,相互之间遗传关系较远。【结论】MD/FJ/F1/08可能是由H6N2、H4N6和H5N1等多亚型AIV基因重组而成。     

我国部分鸡源H9N2亚型流感病毒NS1基因序列分析

对1996年至2001年间自我国部分养鸡场发病鸡或死亡鸡分离鉴定的8株H9N2亚型禽流感病毒的非结构蛋白基因(NS1)进行了扩增和序列测定,并分析和比较了其核苷酸和氨基酸的同源性。结果表明, NS1基因核苷酸和氨基酸同源性分别为96.5%～99.5% 和94.5～98.6%, 说明NS1基因在遗传进化上高度保守,稳定遗传。与中国香港、韩国、巴基斯坦及人源H9N2分离株相比较,发现中国大陆的鸡源H9N2分离株的NS1基因在其羧基端缺少13个氨基酸。系统进化树分析表明,该8株病毒的NS1基因属于相同的进化分支,而且中国的早年分离株A/chicken/Beijing/1/94位于该进化分支的根部,暗示这些分离株的NS1基因是由A/chicken/Beijing/1/94演化而来;尚未发现NS1基因属于A/quail/Hong Kong/G1/97like分支的分离株。同时,系统进化树也说明了我国的H9N2分离株与韩国、巴基斯坦等地的H9N2分离株隶属于不同的进化分支,H9N2亚型禽流感的发生和流行与地域有一定的相关性。     

我国部分鸡源H9N2亚型流感病毒NSl基因序列分析

对1996年至2001年间自我国部分养鸡场发病鸡或死亡鸡分离鉴定的8株H9N2亚型禽流感病毒的非结构蛋白基因(NSl)进行了扩增和序列测定,并分析和比较了其核苷酸和氨基酸的同源性。结果表明：NSI基因核苷酸和氨基酸同源性分别为96．5％-99．5％和94.5-98．6％,说明NSl基因在遗传进化上高度保守,稳定遗传。与中国香港、韩国、巴基斯坦及人源H9N2分离株相比较,发现中国大陆的鸡源H9N2分离株的NSl基因在其羧基端缺少13个氨基酸。系统进化树分析表明,该8株病毒的NSl基因属于相同的进化分支,而且中国的早年分离株A／chicken／Beijing/1／94位于该进化分支的根部,暗示这些分离株的NSl基因是由A/chicken／Beijing/1／94演化而来;尚未发现NSl基因属于A/quail／HongKong／G1／97-1ike分支的分离株。同时,系统进化树也说明了我国的H9N2分离株与韩国、巴基斯坦等地的H9N2分离株隶属于不同的进化分支,H9N2亚型禽流感的发生和流行与地域有一定的相关性。     

禽流感病毒分离株NS基因同源性及等位基因类型分析

目的　克隆测定国内具有代表性的禽流感病毒 (AIV)的非结构 (NS)蛋白基因核苷酸序列 ,分析其同源性和等位基因类型 ,为进一步探索禽流感NS蛋白抗体监测方法奠定基础。方法　经RT PCR扩增了国内 3株H9N2、2株H5N1、2株H7N2亚型AIV分离株的NS蛋白基因 ,并把扩增的基因片段克隆到pGEM T载体中测序 ,将测序结果与GenBank中的核苷酸序列进行同源性比较 ,绘制基因进化树。结果　经测序获得了各AIV分离株NS基因的完整编码序列。同源性分析表明 ,3株H9亚型AIV的NS基因之间的同源性为 96 %～ 98% ;两株H5亚型AIVNS基因同源性为 91 6 % ;两株H7亚型AIV的NS基因同源性为 98 9%。H5和H9亚型分离株的NS基因之间的同源性均高于 90 % ;而H7N2亚型分离株与其它两种亚型分离株的NS基因同源性约为 6 0 %～ 70 %。在AIVNS基因系统发育进化树中 ,H5、H9亚型分离株都处于等位基因A群内 ;3株H9亚型分离株的进化关系较近 ,与香港、广东的部分H5N1病毒株起源相同 ,而 2株H5病毒的NS基因则处于不同分枝内 ;2株H7亚型分离株的NS基因都处于等位基因B群内 ,进化关系较近。结论　这 7株国内AIV分离株的NS基因之间的同源性差异较大 ,约为 6 0 %～ 99% ,且包括A、B两种类型的等位基因     

H3亚型鸭源流感病毒聚合酶PB1基因的序列分析

目的阐明H3亚型鸭流感病毒与其他亚型流感病毒的关系。方法对活禽市场分离的3株H3N8亚型鸭源流感病毒聚合酶PB1基因进行了序列分析。结果3株鸭源H3N8流感病毒聚合酶PB1基因核苷酸同源性为99.9%,与H9N2亚型流感病毒(DK/ST/2143/00)的同源性为96.31%~96.44%,而与H3N8亚型鸭流感病毒(Mal/Alberta/279/98)为88.65%~88.79%。系统进化树分析表明,本实验中的3株病毒属于相同的分支,且与A/duck/Hong Kong/Y439为代表的H9N2亚型禽流感病毒位于一进化分支,说明三株H3N8亚型流感病毒重排了H9N2亚型禽流感病毒的基因片段。结论不同亚型禽流感病毒在贮存宿主体内的重排以及重排病毒的新特点如鸭H3N8亚型流感病毒对禽的致病性,应当引起我们的高度重视。     

H5N1亚型禽流感病毒HA基因核酸序列的变异分析

从GenBank上获得194株不同来源的H5N1亚型禽流感病毒HA基因核酸序列,利用MEGA3分析了HA基N核酸序列碱基突变的特点。并通过比较序列同源性,构建NJ系统进化树,探讨了不同来源的H5N1病毒的系统进化关系。序列分析结果表明,被研究的194条H5N1亚型禽流感病毒HA基因核酸序列,碱基长度大约在1700bp左右,共发现了757个可变位点,其中Parsimony—informative sites有537个,Singleton sites有220个;病毒的变异速率很快,平均变异率为3．23％;病毒的序列变异具有显著的地区特点和时间特点;同时,全球化的贸易以及候鸟的迁徙在传播病毒过程中起一定作用。     

山东地区16株H9N2亚型禽流感病毒HA基因的序列分析

为了解H9N2亚型禽流感病毒(AIV)山东分离株的遗传变异情况,采用RT-PCR技术对16株从山东不同地区分离的H9N2亚型禽流感病毒的HA基因进行扩增、克隆和测序,并对所获得的HA全序列进行同源性和遗传进化分析。结果显示,16个分离株的裂解位点均为RSSR↓GLF,符合低致病性禽流感病毒的分子特征;有7～9个潜在糖基化位点;受体结合位点除198位有变异,其他位点均较保守;234位氨基酸均为L,具有与哺乳动物唾液酸α,2-6受体结合的特征;16个分离株HA基因核苷酸及氨基酸序列同源性分别为96.3%～99.9%和97.1%～99.6%;16个分离株同属于欧亚分支中的A/Duck/Hong Kong/Y280/97亚群。     

H9N2亚型禽流感病毒基因组的遗传进化分析

2009～2011年从江苏省、湖北省和安徽省等地来源于鸡、鸭、鹌鹑和鸽子的样品中分离鉴定出16株H9N2亚型禽流感病毒。通过反转录聚合酶链式反应(RT-PCR)扩增出分离株的全基因片段,并对其进行测序及遗传进化分析。序列分析显示,16株病毒HA基因裂解位点氨基酸序列为P-S-R/K-S-S-R,符合低致病性禽流感的分子特征;226位均为L,具有与哺乳动物唾液酸α,2-6受体结合的特性。M2基因均出现了对金刚烷胺产生耐药性的N31S突变。不同宿主来源的H9亚型AIV的主要分子特征一致。全基因遗传进化分析表明16株H9N2亚型禽流感病毒全基因发生了3配体重组,即以F98亚系AIV为骨架,HA来源于Y280亚系,PB2和M基因来源于G1亚系,形成了2种新的基因型。因此,要加强对H9N2亚型禽流感病毒的监测,密切关注它的重组趋势。     

Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

Migratory aquatic birds play an important role in the maintenance and spread of avian influenza viruses (AIV). Many species of aquatic migratory birds tend to use similar migration routes, also known as flyways, which serve as important circuits for the dissemination of AIV. In recent years there has been extensive surveillance of the virus in aquatic birds in the Northern Hemisphere; however in contrast only a few studies have been attempted to detect AIV in wild birds in South America. There are major flyways connecting South America to Central and North America, whereas avian migration routes between South America and the remaining continents are uncommon. As a result, it has been hypothesized that South American AIV strains would be most closely related to the strains from North America than to those from other regions in the world. We characterized the full genome of three AIV subtype H11N9 isolates obtained from ruddy turnstones (Arenaria interpres) on the Amazon coast of Brazil. For all gene segments, all three strains consistently clustered together within evolutionary lineages of AIV that had been previously described from aquatic birds in North America. In particular, the H11N9 isolates were remarkably closely related to AIV strains from shorebirds sampled at the Delaware Bay region, on the Northeastern coast of the USA, more than 5000 km away from where the isolates were retrieved. Additionally, there was also evidence of genetic similarity to AIV strains from ducks and teals from interior USA and Canada. These findings corroborate that migratory flyways of aquatic birds play an important role in determining the genetic structure of AIV in the Western hemisphere, with a strong epidemiological connectivity between North and South America.     

Influenza A Virus Migration and Persistence in North American Wild Birds

Wild birds have been implicated in the emergence of human and livestock influenza. The successful prediction of viral spread and disease emergence, as well as formulation of preparedness plans have been hampered by a critical lack of knowledge of viral movements between different host populations. The patterns of viral spread and subsequent risk posed by wild bird viruses therefore remain unpredictable. Here we analyze genomic data, including 287 newly sequenced avian influenza A virus (AIV) samples isolated over a 34-year period of continuous systematic surveillance of North American migratory birds. We use a Bayesian statistical framework to test hypotheses of viral migration, population structure and patterns of genetic reassortment. Our results reveal that despite the high prevalence of Charadriiformes infected in Delaware Bay this host population does not appear to significantly contribute to the North American AIV diversity sampled in Anseriformes. In contrast, influenza viruses sampled from Anseriformes in Alberta are representative of the AIV diversity circulating in North American Anseriformes. While AIV may be restricted to specific migratory flyways over short time frames, our large-scale analysis showed that the long-term persistence of AIV was independent of bird flyways with migration between populations throughout North America. Analysis of long-term surveillance data provides vital insights to develop appropriately informed predictive models critical for pandemic preparedness and livestock protection.     

The evolutionary genetics and emergence of avian influenza viruses in wild birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient "genome constellations," continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.     

环渤海滨海湿地鸻鹬类水鸟多样性及其环境影响因子

环渤海湿地是水鸟南北迁徙的重要驿站,尤其对于该线路上的鸻鹬鸟类具有非常重要的意义。以环渤海地区12处典型滨海湿地为研究对象,于2016-2020年每年春季开展水鸟调查,明确了鸻鹬类水鸟群落组成及其时空变化,采用结构方程模型 （Structural Equation Modeling,SEM）分析了鸻鹬类水鸟多样性与环境因子的响应关系,评估了各环境因子的影响强度。结果表明：（1）共记录到鸻鹬类水鸟7科51种,几乎全部为旅鸟。全球极危物种1种,濒危物种3种,近危物种9种。国家一级保护鸟类2种,国家二级保护鸟类8种。黑腹滨鹬（Calidris alpina）、大滨鹬（Calidris tenuirostris）、黑尾塍鹬（Limosa limosa）、灰鸻（Pluvialis squatarola）、斑尾塍鹬（Limosa lapponica）个体数量最多。（2）山东黄河三角洲、辽宁辽河口、天津北大港等河口湿地,水鸟种类多,单位面积水鸟数量较少。（3）河北沧州沿海、山东滨州贝壳堤岛及其周边区域为环渤海地区湿地集中区,水鸟种类较多。（4）综合影响强度为保护强度>食物>气候,建立自然保护地是保护水鸟多样性的最有效措施。（5）建议将河北南大港湿地和鸟类省级自然保护区提升至国家级,扩大滨州贝壳堤岛与湿地国家级自然保护区面积,对山东黄河三角洲、辽宁辽河口覆盖的各级各类自然保护地进行优化整合。研究结果能为环渤海地区鸻鹬类水鸟保护策略的制定提供相关依据。     

Avian Influenza Virus Surveillance in Wild Birds in Georgia: 2009–2011

The Caucasus, at the border of Europe and Asia, is important for migration and over-wintering of wild waterbirds. Three flyways, the Central Asian, East Africa-West Asia, and Mediterranean/Black Sea flyways, converge in the Caucasus region. Thus, the Caucasus region might act as a migratory bridge for influenza virus transmission when birds aggregate in high concentrations in the post-breeding, migrating and overwintering periods. Since August 2009, we have established a surveillance network for influenza viruses in wild birds, using five sample areas geographically spread throughout suitable habitats in both eastern and western Georgia. We took paired tracheal and cloacal swabs and fresh feces samples. We collected 8343 swabs from 76 species belonging to 17 families in 11 orders of birds, of which 84 were real-time RT-PCR positive for avian influenza virus (AIV). No highly pathogenic AIV (HPAIV) H5 or H7 viruses were detected. The overall AIV prevalence was 1.6%. We observed peak prevalence in large gulls during the autumn migration (5.3–9.8%), but peak prevalence in Black-headed Gulls in spring (4.2–13%). In ducks, we observed increased AIV prevalence during the autumn post-moult aggregations and migration stop-over period (6.3%) but at lower levels to those observed in other more northerly post-moult areas in Eurasia. We observed another prevalence peak in the overwintering period (0.14–5.9%). Serological and virological monitoring of a breeding colony of Armenian Gulls showed that adult birds were seropositive on arrival at the breeding colony, but juveniles remained serologically and virologically negative for AIV throughout their time on the breeding grounds, in contrast to gull AIV data from other geographic regions. We show that close phylogenetic relatives of viruses isolated in Georgia are sourced from a wide geographic area throughout Western and Central Eurasia, and from areas that are represented by multiple different flyways, likely linking different host sub-populations.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Complete Genome Sequence of an H9N2 Avian Influenza Virus Isolated from Egret in Lake Dongting Wetland

We isolated a recombinant H9N2 avian influenza virus (AIV) from fresh egret feces in the Ardeidae protection region of the Dongting Lake wetland area in China, and it was designated A/Egret/Hunan/1/2012(H9N2). This is the first report of isolating H9N2 AIV from wild birds in the Dongting Lake wetland. Its eight gene segments are generated by reassortment of gene segments of different AIV subtypes. These results are helpful for understanding the epidemiology and evolution of AIV in wild birds during migration.     

Influenza a viruses from wild birds in Guatemala belong to the North American lineage

The role wild bird species play in the transmission and ecology of avian influenza virus (AIV) is well established; however, there are significant gaps in our understanding of the worldwide distribution of these viruses, specifically about the prevalence and/or significance of AIV in Central and South America. As part of an assessment of the ecology of AIV in Guatemala, we conducted active surveillance in wild birds on the Pacific and Atlantic coasts. Cloacal and tracheal swab samples taken from resident and migratory wild birds were collected from February 2007 to January 2010.1913 samples were collected and virus was detected by real time RT-PCR (rRT-PCR) in 28 swab samples from ducks (Anas discors). Virus isolation was attempted for these positive samples, and 15 isolates were obtained from the migratory duck species Blue-winged teal. The subtypes identified included H7N9, H11N2, H3N8, H5N3, H8N4, and H5N4. Phylogenetic analysis of the viral sequences revealed that AIV isolates are highly similar to viruses from the North American lineage suggesting that bird migration dictates the ecology of these viruses in the Guatemalan bird population.     

Is the occurrence of avian influenza virus in Charadriiformes species and location dependent?

Birds in the order Charadriiformes were sampled at multiple sites in the eastern half of the continental USA, as well as at Argentina, Chile, and Bermuda, during 1999-2005, and tested for avian influenza virus (AIV). Of more than 9,400 birds sampled, AIV virus was isolated from 290 birds. Although Ruddy Turnstones (Arenaria interpres) comprised just 25% of birds sampled, they accounted for 87% of isolates. Only eight AIV isolations were made from birds at four locations outside of the Delaware Bay, USA, region; six of these were from gulls (Laridae). At Delaware Bay, AIV isolations were predominated by hemagglutinin (HA) subtype H10, but subtype diversity varied each year. These results suggest that AIV infection among shorebirds (Scolopacidae) may be localized, species specific, and highly variable in relation to AIV subtype diversity.     

Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City,China

Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response.