猪流感病毒NA和NS1基因部分区段的基因功能定位研究

项目名称： 猪流感病毒NA和NS1基因部分区段的基因功能定位研究

项目编号： No.30800832

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 周红波

作者单位： 华中农业大学

项目金额： 20万元

中文摘要： 由猪流感病毒引起的猪流感是危害当前养猪业的重要疫病。猪流感病毒NA 基因在进化过程中选择不同的“#33550;部-Motif”#23545;HA-NA 活性平衡、病毒复制和致病性等有何影响，目前还不得而知。NS1真核转录启动因子（eIF4GI）结合区内5 氨基酸自然缺失的生物学意义及eIF4GI 结合区进一步缺失截短对猪流感病毒分子进化、复制及致病性的影响值得深入研究。本项目利用反向遗传技术成功构建了流感病毒NA及NS1突变株，对突变株生物学特性、毒力、致病性进行了全面评价。课题进展顺利，超额完成课题任务书规定的任务。获得了9株不同NA“#33550;部-Motif”#21644;5株NS1eIF4GI结合区突变的重配病毒，证实NA基因茎部出现的第49-68位共20个氨基酸缺失的特殊的“#33550;部-Motif”可能是2000年后H5N1病毒毒力和致病性增强的一个重要因素；发现NS1 蛋白5个氨基酸的缺失可提高病毒对鸡和小鼠的毒力和致病性，NS1 eIF4GI结合区的缺失导致病毒毒力显著致弱，降低了病毒抑制宿主干扰素产生的能力。揭示了流感病毒致病的分子机制。发表标注本项目资助SCI论文7篇，获国家科技进步二等奖1项，培养硕士研究生3名。

中文关键词： 猪流感病毒；反向遗传操作技术；基因功能定位；分子机制

英文摘要： Swine influenza, caused by swine influenza virus, is an important infectious disease in swine industry. However, the biologic characteristic of NA stalk-motif and whether NAs with different stalk-motifs caused the difference of virulence and pathogenesis in H5N1 influenza virus had not yet been studied in detail. There was a five amino acids deletion at the position 80-84 of NS1 eIF4GI binding domain in H5N1 isolates after 2000. The biologic role of these five amino acids deletion and whether the shortenings of NS1 eIF4GI binding domain influenced the evolution, replication and pathogenesis of swine influenza virus had not yet been studied in depth. In this study, the site-specific mutagenesis, deletion and insertion have been done in the NA stalk region and NS1 eIF4GI binding domain of H5N1 influenza virus and the recombinant mutants were generated by the reverse genetics operation. We have located the functional gene regions correlated with virus reproduction, replication, pathogenesis and immune escape of swine influenza virus by determining and comparing these mutants. These indicate that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The NS1 eIF4GI binding domain-truncated mutants were attenuated in replication in vitro and in vivo, and that the level of attenuation correlated with the amounts of IFN-βnduced by the respective NS1-truncated viruses. Based on above, we revealed the molecular pathogenesis mechanism of swine influenza virus. In this project, 7 SCI papers have been published. Three postgraduate have graduated for HZAU by granted NSFC NO. 30800832.

英文关键词： Swine influenza virus; Reverse genetics operation; Gene functional localization; Molecular mechanism