野生鸟类高致病性禽流感抗体水平检测方法研究

高致病性禽流感是A型流感病毒引起的一种禽类毁灭性疾病．从2004年1月27日我国公布首例确诊高致病性禽流感疫情以来,截止2004年2月4日,已出现高致病性禽流感疫情23例, 其中疑似18例,确诊5例;全国合计病禽56 417只,死亡49 236只,扑杀1 215 057只．H5N1亚型毒株除致鸡大量发病死亡外,还会使雉鸡科、鸭科、鸠鸽科和鹭科等野生鸟类致病．2005年5月4日, 在青海省刚察县泉吉乡年乃索麻村部分候鸟发生死亡,国家禽流感参考实验室5月18日从死鸟体内分离到H5N1亚型禽流感病毒．截止5月26日,仅青海省已有包括斑头雁、鱼鸥等在内的超过 1 000只候鸟死亡．自从2004年1月以来,全球因感染禽流感疫病,已造成了近百人死亡．研究结果表明,野生鸟类的迁徙,是造成疫病传播蔓延最主要的途径．西安市在2004年2月,对全市养殖的野生鸟类进行了禽流感疫苗接种,2005年的免疫工作即将展开,但目前在全国尚无野生鸟类疫苗接种注射剂量和方法的规范,已经接种过的野生鸟类是否具有禽流感抗体及产生抗体的效价还不清楚,更为严峻的是至今还没有可行的野生鸟类抗体水平检测的方法．所以,进行野生鸟类高致病性禽流感抗体水平检测方法课题研究,并为研究野生鸟类产生有效抗体的疫苗接种注射剂量,可为防控本病提供科学依据．     

湖北万朝山鸟类物种资源调查

通过野外观察统计、收集历史资料、座谈访问等方法，对万朝山鸟类多样性进行了调查，确认万朝山自然保护区鸟类140种，隶属于12目，34科．其中，古北种41种，东洋种76种，广布种23种；国家Ⅰ级野生保护鸟类1种，Ⅱ级39种Ⅰ被列入《濒危野生动植物种国际贸易公约》的鸟类38种（附录Ⅰ1种，附录Ⅱ35种，附录Ⅲ2种）1分析了鸟类种类组成、特点、季节型、区系组成、鸟类价值；提出了鸟类保护的建议。     

武汉市区及市郊鸟类多样性初步调查

鸟类多样性变化可以反映环境变化．运用样线法和样点法，于2000年9月至2004年6月对武汉市区及市郊鸟类种类组成和多样性进行了调查．结果共记录鸟类127种，分属于16目49科91属．区系成分具有南北混杂的特点．其Shannon-Wiener指数为6．277，G指数为4．351，F指数为20．261，G-F指数为0、785．各生境类型的多样性指数都较低，但均匀性指数都较高，各生境间的相似性指数较低，比较而言，农田（5．357）和树林（5．737）中鸟类多样性相对较高，农田（0．903）鸟类均匀性指数最高，树林与园林住宅之间相似性系数（0．821）最高．大量候鸟聚集武汉，是研究候鸟迁徙和禽流感等禽源性疾病防控的重要区域．     

南充高坪机场春季鸟类群落研究

2006年3-5月，通过对南充高坪机场及附近5种生境6条样带的调查，共记录春季鸟类67种，分属11目35科．其中东洋种28种（41．8％）；古北种27种（40．3％）；广布种3种（4．5％）；不易归类的种类9种（13．4％）．繁殖鸟类中，东洋种28种（62．2％）；古北种14种（31．1％）．夏候鸟25种（37．3％）；冬候鸟10种（14．9％）；留鸟28种（41．8％）；旅鸟4种（6．0％）．通过重要性值分析表明，最重要的鸟11种，有家燕，金腰燕、白鹭、棕背伯劳、白颊噪鹛、家鸽等．按鸟类的生境分布系数划分，广性分布型鸟类是31种．优势种（D〉10）3种，分别是家燕，金腰燕，白鹭．     

网湖湿地自然保护区鸟类初步研究

2003年-2004年对湖北阳新网湖湿地自然保护区鸟类资源进行了调查，共记录到鸟类173种，隶属于15目38科．对鸟类的区系组成、居留型及重点保护鸟类进行研究，发现当地有繁殖鸟类104种（夏候鸟42种、留鸟62种），非繁殖鸟类69种（冬候鸟51种、旅鸟18种）；古北种88种，东洋种62种，广布种23种；国家重点保护野生动物31种（其中，国家Ⅰ级保护动物3种，国家Ⅱ级的有28种），有湖北省重点保护鸟类47种，被列入《濒危野生动植物种国际贸易公约》的鸟类有30种，被列入《中国濒危动物红皮书》的鸟类有12种．对鸟类的保护提出了存在的问题和建议．     

全球禽流感考验中国创新力——禽流感的科学防控进路

禽流感病毒通过迁徙鸟类的携带传播，表明禽流感病毒具有在短时间内迅速演化为全球危机的可能，因此，任何小范围的局部病毒传播，都应当引起世界各国的一致重视。[编按]     

禽流感研究进展:迁徙鸟的作用与反向遗传技术的贡献

禽流感疫情在世界范围内的频繁暴发不仅给家禽业生产带来了巨大的损失,也给人类的生命安全造成了严重威胁。自2005年青海湖暴发大规模野生鸟类感染高致病性禽流感病毒事件以来,候鸟因作为传播病毒的“载体”而备受关注。与此同时,针对禽流感跨种间传播及致病机制、禽流感防治等研究也取得了重要进展。本文重点综述了候鸟的迁徙在禽流感传播中的作用以及反向遗传技术在禽流感研究中做出的重要贡献。     

人乳头瘤病毒基因分型检测在宫颈癌早期筛查中的应用

目的探讨女性生殖道感染人乳头瘤病毒（HPV）基因检测与颈癌前病变的关系。方法采用HPV分型基因检测系统对妇科门诊就诊的妇女进行宫颈细胞HPV DNA检测（同时检测5种低危型和18种高危型HPV亚型）。结果827例标本中共检出HPV阳性210例（25．4％）,共检出20种HPV亚型,其中单纯低危型HPV亚型感染45例（5．4％）,单纯高危型HPV亚型感染142例（17．2％）,高危、低危亚型共同感染13例（1．6％）;检出感染单一HPV亚型187例（22．6％）,感染两种亚型以上23例（2．8％）。低危型主要为HPV11,其次为HPV43、6;高危型主要为HPV16、其次为58、31、33、18等亚型。结论女性生殖道HPV感染者较易发生颈癌前病变,提示早期发现及防治HPV感染对预防子宫颈癌的发生具有重要意义。     

PCR扩增检测献血者中TT病毒DNA及部分TTV基因序列分析

目的：了解江苏地区献血者中新型肝炎相关病毒－－TT病毒的感染状况，探讨TTV感染与ALT异常的相关性，方法：设计通用引物，采用半套式聚合酶链反应（hemi-nested PCR)方法检测195份献血者血清标本，结果，在血清丙氨酸转氨酶（ALT）异常，HBsAg及抗HCV阴性的99份献血者血清标本中，检出TTV DNA阳性标本36份，TTV DNA的阳性检出率为36．3％，而在96份正常献血者血清标本中，检出TTV DNA阳性标本16份，TTV DNA阳性检出率为16．6％，明显低于ALT常献血者人群，对2株阳性株序列分析结果显示，一株与TX011（G1b日本代表株）的同源性为98．6％，属于G1b亚型，另一株虽可归属于G1，但与G1a ，Blb差异较大，可能为G1的新亚型，结论：江苏地区献血者人群中存在TTV感染，国内首次报告检出TTV G1b亚型及新的亚型，献血者ALT异常与TTV感染密切相关，输血可能成为传播TTV感染的途径之一。     

“备战”H7N9禽流感

近期,H7N9禽流感疫情是人们普遍关注的事件。从3月底上海首次公布,确定发现人感染H7N9禽流感并出现死亡病例后,人们谈之色变。"备战"H7N9禽流感,成为大家的重要任务。H7N9禽流感H7病毒通常是一组在鸟类中传播的流感病毒,H7N9是H7病毒大类下的一个亚群。     

Molecular patterns of avian influenza A viruses

Avian influenza A viruses could get across the species barrier and be fatal to humans. Highly pathogenic avian influenza H5N1 virus was an example. The mechanism of interspecies transmission is not clear as yet. In this research, the protein sequences of 237 influenza A viruses with different subtypes were transformed into pseudo-signals. The energy features were extracted by the method of wavelet packet decomposition and used for virus classification by the method of hierarchical clustering. The clustering results showed that five patterns existed in avian influenza A viruses, which associated with the phenotype of interspecies transmission, and that avian viruses with patterns C and E could across species barrier and those with patterns A, B and D might not have the abilities. The results could be used to construct an early warning system to predict the transmissibility of avian influenza A viruses to humans.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

禽流感疫苗的研究进展

近年来禽流感的大范围流行,带来了巨大的经济损失并严重威胁人类的健康.由于其重要的经济和公共卫生学意义,使得禽流感的防制显得突出重要.疫苗的使用是控制禽流感的主要手段.目前实际应用中的以禽流感全毒灭活苗为主,但其由于潜在的缺点使得人们将目光转向其它类型的疫苗的研制.本文就目前禽流感疫苗的研究方面介绍了国内外的一些研究进展.     

禽流感流行与防制现状

禽流感是禽的烈性传染病，对养禽业造成严重危害，同时威胁着人类的健康．本文对国内外禽流感近年来的流行情况、禽流感病毒的特征及危害、临床症状和病理变化、禽流感的诊断技术和防治进行了综述．     

H5亚型禽流感病毒HA基因昆虫/杆状病毒系统的表达及对BALB/c小鼠的免疫保护

经RT-PCR扩增了禽流感病毒A/Goose/Guangdong/1／96 H5N1亚型1．7kb HA基因的cDNA,将其克隆到pMD18-T中并测序。亚克隆到杆状病毒转移载体pMelBacA的蜜蜂蜂毒素分泌信号下游中,测序正确后与线性化的杆状病毒DNA(Bac-N-BlueTM DNA)共转染Sf9昆虫细胞。将重组杆状病毒感染HFive细胞,72h左右收获细胞,超声波裂解,SDS—PAGE结果表明HA基因在重组杆状病毒感染的HFive细胞中获得表达。蛋白胶薄层扫描分析显示:表达的HA蛋白占重组杆状病毒感染细胞总蛋白含量的17．1％。Western-blot 及血凝实验结果显示,表达的禽流感H5N1亚型病毒HA蛋白具有生物学活性。表达的H5 HA蛋白定量乳化后,皮下多点注射免疫SPF 级BALB/c雌性小鼠,免疫后产生了H5 HA特异抗体,并在三免前后达到并保持较高水平。用致死剂量的HPAIV H5N1攻击小鼠,免疫组小鼠提供了100％的保护力,而对照组小鼠先后发病且死亡:为研制禽流感H5N1亚型病毒亚单位疫苗,防制禽流感奠定了基础。     

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.