禽流感病毒三种检测方法的比较研究

对分别应用病毒分离鉴定、反转录-聚合酶链式反应(RT-PCR)、间接免疫荧光(IFA)3种方法对病料组织中的禽流感病毒(AIV) 进行检测与比较研究。结果显示:经鸡胚尿囊腔接种分离到的病毒,通过用禽流感病毒H5、H9亚型单克隆抗体所进行的血凝(HA)和血凝抑制试验(HI),鉴定为AIV H5亚型毒株;应用RT-PCR技术直接对病料组织抽提的RNA样品进行检测,从病料组织能扩增出大小为229bp的AIV通用目的片段和大小为380bp的H5亚型特异性片段;取病料组织的过滤液接种狗肾上皮细胞(MDCK)单层,24h后用H5亚型AIV特异性的单克隆抗体进行间接免疫荧光试验,结果在细胞核、细胞质内观察到特异性的荧光。研究的结果表明,3种技术都可对病料样品中的AIV进行检测,而RT-PCR和IFA两种方法还可直接对病料样品中的病毒进行亚型的鉴定。相对而言,后两者具有更快速、更敏感、操作更简便的特点。     

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.     

A型流感病毒基质蛋白M1的二聚化机制

流感病毒基质蛋白1(matrix protein 1,M1)位于病毒包膜之下,形成一层壳状结构(衣壳),可与病毒的血凝素、神经氨酸酶、包膜和病毒遗传物质发生相互作用,在病毒的组装与复制过程中起着关键作用.不过,除N端有晶体结构外,全长M1蛋白的结构尚未解析.因此,人们对全长M1蛋白如何二聚化、然后多聚化形成病毒衣壳的过程知之甚少.为了解M1蛋白的二聚化机制,首先从M1的N端片段晶体结构出发,用蛋白质结构预测方法获得M1的全长结构模型;其次,对可能的M1二聚体进行分子动力学模拟,分析其二聚化界面的氨基酸,由此发现了一种通过M1蛋白C端片段结合而形成的二聚体;最后,为验证模拟结果,用电镜三维重构方法分析了全长M1二聚体的构象,并提出了M1多聚化的机理模型.     

甲型流感病毒PB1-PA蛋白作用位点的发现与分析

 基于共进化理论,探究了甲型流感病毒PB1蛋白与PA蛋白上具有共同进化可能性的保守九聚片段 (C9MP)。结构信息显示PB1蛋白的第1-15位氨基酸与PA蛋白的第239-716位氨基酸具有相互作用域;对该区域变异分布的分析发现,PA蛋白第670位氨基酸Q所在的C9MP与PB1蛋白的第9位氨基酸F、第12位氨基酸V和第13位氨基酸P所在的C9MP在PB1-MP1相互作用面上具有最低的共进化值。结合DSSP程序的分析表明,由PA蛋白第670位氨基酸Q与PB1蛋白的第9位氨基酸F、第12位氨基酸V与第13位氨基酸P构成的区域可能成为潜在的相互作用位点。     

Influenza A （HIN1） transmission by road traffic between cities and towns

Influenza A (H1N1) was spread widely between cities and towns by road traffic and had a major impact on public health in China in 2009. Understanding regulation of its transmission is of great significance with urbanization ongoing and for mitigation of damage by the epidemic. We analyzed influenza A (H1N1) spatiotemporal transmission and risk factors along roads in Changsha, and combined diffusion velocity and floating population size to construct an epidemic diffusion model to simulate its transmission between cities and towns. The results showed that areas along the highways and road intersections had a higher incidence rate than other areas. Expressways and county roads played an important role in the rapid development stage and the epidemic peak, respectively, and intercity bus stations showed a high risk of disease transmission. The model simulates the intensity and center of disease outbreaks in cities and towns, and provides a more complete simulation of the disease spatiotemporal process than other models.     

Special features of the 2009 pandemic swine-origin influenza A H1N1 hemagglutinin and neuraminidase

Since the 2009 pandemic H1N1 swine-origin influenza A virus (09 S-OIV) has reminded the world about the global threat of the ever changing influenza virus,many questions regarding the detailed re-assortment of influenza viruses yet remain unanswered.Influenza A virus is the causative agent of the pandemic flu and contains 2 major antigenic glycoproteins on its surface:(i) hemagglutinin (HA);and (ii) neuraminidase (NA).The structures of the 09 S-OIV HA and NA proteins (09H1 and 09N1) have recently been resolved in our laboratory and provide some clues as to why the 09 S-OIV re-assortment virus is highly infectious with severe consequences in humans.For example,the 09H1 is highly similar to the HA of the 1918 influenza A pandemic virus in overall structure and especially in regards to its 5 defined antibody binding epitopes.For 09N1,its most distinctive feature is the lack of a 150-loop active site cavity,which was previously predicted to be present in all N1 NAs,and we hypothesize that the 150-loop may play a important role in the substrate specificity (α2,3 or α2,6 linked sialic acid receptors) and enzymatic mechanism of influenza NA.Combination of the HA and NA with special characteristics for the 09 S-OIV might contribute to its high increased transmissibility in humans.     

单价流感疫苗脂质体干粉细胞免疫研究

从细胞免疫水平考察流感疫苗脂质体干粉肺部免疫的免疫原性,以验证在稳定性提高的同时,流感疫苗脂质体干粉肺部免疫原性不低于现行应用的流感疫苗原液腹腔注射免疫。将实验小鼠分为2个大组,每组分为阴性对照组、疫苗脂质体冻干粉组、非脂质体流感疫苗原液组和阳性对照组(n=5)。非脂质体流感疫苗原液组和疫苗脂质体冻干粉组分别以每只6μg血凝素(以H1N1计)肺部灌注免疫,同时以每只6μg非脂质体流感疫苗原液组腹腔免疫作为阳性对照。分别免疫14 d和28 d后,用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测脾淋巴细胞增殖情况,以考察其细胞免疫原性。脂质体肺部免疫可以诱导细胞免疫,且其免疫原性明显高于流感疫苗原液传统腹腔注射免疫组。与流感疫苗原液腹腔注射免疫相比,流感疫苗脂质体干粉通过肺部免疫,细胞免疫效果明显提高。     

鸭禽流感母源抗体的消长规律及其免疫效果

为研究鸭禽流感免疫程序,对不同日龄的雏鸭进行禽流感母源抗体的检测,并对雏鸭接种禽流感H5、H9二价油乳剂苗后的HI抗体水平进行了检测。结果表明,雏鸭的母源抗体水平在1日龄达最高值,然后逐渐下降;H5抗体20日龄后降低到4.5log2以下,H9抗体25日龄后降低到4.8log2以下。根据试验采用的免疫方案,对雏鸭在8日龄时进行首免,并多次免疫后可提高H5、H9抗体水平,有效预防禽流感的发生。     

基于AGENT的流感传播模型与分析——以香港流感模式为例

应用基于主体的建模(ABM)方法,建立了流感病毒的多传播路径模型,并依此对香港地区的流感传播的动态模式进行分析,得到结果:1)香港地区流感传播的夏季高峰因为病毒通过污染物的传播而产生;2)在绝对湿度的影响下,流感病毒的空气传播途径和污物传播途径分别在冬季和夏季起主导作用;3)两种传播路径的模型能够合理地解释香港地区流感双峰现象产生的原因,为在不同季节制定有针对性的防控措施提供理论依据。     

A/PR8/34(H1N1)NA基因包装信号对H5N1流感疫苗株NIBRG-14产量的影响

NIBRG-14是采用"6+2"策略制备的一株H5N1灭活疫苗株,其表面抗原HA和NA基因来自于A/Vietnam/1194/2004(H5N1,VN1194),内部基因来自于A/Puerto Rico/8/34(H1N1,PR8),已有研究表明该疫苗株在鸡胚中的产量不佳.本研究发现,在PR8背景下,VN1194NA基因被包装入重组病毒中的效率仅为正常包装量的38%～68%,因此有一部分重组病毒为不含有NAvRNA的缺陷型病毒粒子.本研究通过在VN1194NA基因完整编码区(CDS)的5′和3′两端嵌合PR8NA基因包装信号序列(vRNA3′末端41bp,5′末端67bp)的方法,使重组病毒中NAvRNA的包装效率得到完全恢复,并且病毒在鸡胚的生长滴度提高了10倍,血凝素HA含量提高了约2·7倍,从而为H5N1流感疫苗株的研制提供了新的思考方向.