Objective To establish an A/H6N1 subtype avian influenza virus reverse genetic system and evaluate the pathogenicity of PB2 E627K in A/H6N1 avian influenza virus, and to explore its molecular basis. Methods rA/ H6N1 and rA/H6N1-627 were rescued by A/Mallard/Sanjiang /275/2007 strain reverse genetic system and site mutation technology. 101EID50 - 106EID50 rA/H6Nl and rA/H6N1-627 were inoculated to the mice separately. The pathogenic data were collected and evaluated by body weight changes, MLD50 and virus titration. Results A/H6N1 subtype avian influenza virus reverse genetic system was successfully established. The results of sequence analysis showed that 8 gene segments of rA/H6Nl were derived from the genome of A/H6N1 and had no nucleic acid changes. rA/H6Nl efficiently infected the mice but were not lethal to them, showing a low pathogenicity in mice ( MLD50 > 106.5EID50). The distribution of virus and the virus titer showed no significant difference with that of A/H6N1 , so the biological characteristics kept the same with A/ H6N1. rA/H6N1-627 could infect mice, with body weight loss, but were not lethal for all the mice in the 106 EID50 group. Conclusions The results show that though PB2-E627K plays important roles in H5N1 influenza, it is not the determining factor in the virulence of A/H6N1 influenza virus. The generation of reverse genetic system and site mutation technology provide foundations for further studies on the pathogenic mechanism, transmission mechanism and virus gene function of A/ H6N1 AIV, also opens up new ways for the development of A/H6N1 AIV vaccines.%目的 利用A/H6N1亚型禽流感病毒的反向遗传平台,评估PB2 E627K对A/H6N1亚型禽流感病毒的致病性,探究A/H6N1流感病毒的致病性分子基础.方法 通过A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007株反向遗传操作系统和点突变技术拯救病毒rA/H6N1和PB2 E627K位点发生突变的rA/H6N1-627,两株拯救病毒分别以101EID50～106EID50的攻毒剂量人工感染BALB/c小鼠,通过体重变化、死亡率、病毒滴定等方面进行致病性分析.结果 成功构建A/H6N1亚型禽流感病毒的反向遗传平台,rA/H6N1的8个基因片段完全源于A/H6N1的基因组,核苷酸序列及生物学特性与A/H6N1完全一致.rA/H6N1能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠呈现低致病性(MLD50＞ 106.5EID50),病毒在小鼠体内的分布情况及各个脏器中的病毒滴度与A/H6N1保持一致；rA/H6N1-627能感染小鼠,引起小鼠体重下降,但不能引起所有106 EID50组小鼠死亡,病毒能在小鼠的肺脏和脑部进行增殖.结论 实验结果表明,在H5N1禽流感中发挥重要作用的PB2-E627K位点并非A/H6N1流感病毒的毒力决定因子.A/H6N1流感病毒致病性的分子基础还有待继续研究,该反向遗传操作系统和点突变技术的建立为研究该亚型流感病毒致病机制、传播机制及病毒基因功能奠定了基础,同时也为A/H6N1亚型禽流感病毒新型疫苗的研制开辟了新途径.
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