促进禽流感病毒变异的因素

The pathogenicity, interspecies transmission, antigenic variation and drug resistance mutation of avian influenza viruses (AIVs) are highly related with their mutation rates. The mutation rates of AIVs vary dramatically among the subtypes, even among the virus isolates of the same subtype. The AIVs with high mutation rates are more dangerous and prone to interspecies transmission, antigenic variation and drug resistance. However, little is known about the determinants which may promote the mutation of AIVs. To explore viral mutation-promoting factors, the reverse genetics technology of influenza virus will be used to develop HA-deleted GFP-expressing reassortant and single-site-mutation viruses. These viruses will be used to infected HA-expressing cells (MDCK-HA2) and the sequences of GFP and NS1 genes will be determined by deep sequencing, or by construction of their cDNA libraries and partial sequence analysis. The mutation rate of each recombinant virus will be calculated and the viral mutation-promoting factors of AIVs will be clarified. The adaptability of the recombinant viruses with mutation-promoting factors will be tested on mice to prove their high mutation rate. On the other hand, a GFP-expressing H9N2 influenza virus will be used to infected different cells and different animal tissues. The mutation rate of the virus in different hosts will be calculated for exploration of the host's mutation-promoting factors. These knowledge about viral and host's mutation-promoting factors of AIVs will provide new insight into AIV evolution.

禽流感病毒的致病性、跨宿主传播、抗原变异、耐药性产生与其变异速率息息相关。不同亚型禽流感病毒的变异速率差异很大、甚至相同亚型不同毒株的变异速率也不尽相同，变异快的禽流感病毒造成的危害更严重,更容易出现跨宿主传播、抗原变异和耐药性，目前还不清楚哪些因素可以加速禽流感的变异。本项目将利用流感病毒反向遗传操作技术，拯救一系列表达GFP的HA缺失病毒，用这些病毒感染表达HA蛋白的MDCK-HA2细胞系，利用cDNA文库测序和深度测序方法确定病毒携带的GFP和NS1基因序列，计算病毒在MDCK-HA2细胞上的变异速率, 明确重组、点突变等在促进禽流感病毒加速变异中的作用，通过小鼠实验验证这些因素是否真正能够促进禽流感病毒适应性突变；此外，利用表达GFP蛋白的H9N2病毒感染不同细胞或动物组织来测定宿主因素对病毒变异的影响。确定促进禽流感病毒变异的主要因素，将从新的角度进一步认识禽流感病毒进化规律。

禽流感病毒的致病性、跨宿主传播、抗原变异、耐药性产生与其变异速率息息相关。不同亚型禽流感病毒的变异速率差异很大、甚至相同亚型不同毒株的变异速率也不尽相同，变异快的禽流感病毒造成的危害更严重，更容易出现跨宿主传播、抗原变异和耐药性。本项目利用流感病毒反向遗传操作技术，拯救一系列重组病毒，将这些病毒在MDCK细胞和小鼠上传代后，利用高通量测序方法确定病毒HA、M和NS1基因序列，计算病毒在MDCK细胞和小鼠体内的变异速率；比较分析1998-2012年间H9N2亚型禽流感病毒的生物学特性，利用反向遗传操作技术和动物实验，探寻H9N2亚型禽流感病毒适应新宿主的关键氨基酸位点；利用表达GFP蛋白的H9N2病毒感染不同细胞或动物组织来测定宿主因素对病毒变异的影响，确定促进禽流感病毒变异的主要宿主因素。本项目发现了H9N2亚型禽流感病毒的PB1基因重组到人源流感病毒后，在小鼠体内变异速率明显提高；解释了引起数次人类大流感爆发时的重组病毒中往往有禽流感病毒PB1参与的生物学意义。发现HA上190位氨基酸与禽流感病毒在小鼠肺部的增殖能力密切相关，进而有利于禽流感病毒发生适应性突变，该研究发现了与跨宿主密切相关的新的氨基酸位点，为监控禽流感病毒能否感染哺乳动物提供重要的靶点。确定了禽流感病毒基因在鸡和小鼠肺脏中易发生A→G的碱基转换，而且在鸡的气管中H9N2亚型禽流感病毒更容易突变，为H9N2亚型禽流感病毒新型疫苗研制提供了理论支持。