H9N2亚型禽流感病毒PB2蛋白哺乳动物适应性变异的筛选与功能鉴定

Identification and Characterization of Mammalian Adaptive Mutations in PB2 Protein of H9N2 Subtype Avian Influenza Virus

研究旨在鉴定H9N2亚型禽流感病毒PB2基因进化产生的哺乳动物适应性突变并明确其在哺乳动物宿主中的复制能力和致病性。试验通过建立中国源H9N2亚型禽流感病毒PB2氨基酸序列库、利用多态性分析以及聚合酶活性检测,筛选出PB2基因演化出的潜在适应性位点,并进一步利用哺乳动物细胞以及小鼠模型明确新突变的哺乳动物适应性。结果显示:PB2氨基酸序列中存在12个具有明显多态性的位点,其中PB2-340K、PB2-588V以及PB2-627V的变化趋势与自2015年起人感染病例增加呈正相关,并表现明显的人宿主偏好;这3个突变中,PB2-E627V显著增强了H9N2亚型禽流感病毒在人源293T细胞中聚合酶活性,并且显著提高了H9N2亚型禽流感病毒在哺乳动物细胞中的复制能力以及在小鼠中的病毒载量和致病性。以上研究表明,H9N2亚型禽流感病毒PB2蛋白表现出哺乳动物适应性进化特点,并且能够增强对小鼠的致病性,带来跨宿主感染风险。

This study aimed to identify the mammalian-adaptive mutations evolved in PB2 gene of H9N2 subtype avian influenza virus and determine their replication and pathogenicity in mammalian hosts.By establishing an amino acid sequence database of PB2 proteins of H9N2 subtype avian influenza viruses isolated from China,and performing polymorphism analysis and polymerase activity assays,potential adaptive mutations in PB2 protein were identified.The mammalian adaptation mutation was further characterized using mammalian cells and a mouse model.The results showed 12 sites with significant polymorphisms in PB2 amino acid sequence.Among these,the changes in PB2-340K,PB2-588V and PB2-627V were positively correlated with the increase in human infection cases after 2015,demonstrating a clear preference for human hosts.Of the three mutations,the PB2-E627V mutation significantly enhanced the polymerase activity of H9N2 subtype avian influenza virus in human 293T cells,significantly increased the viral replication in mammalian cells,and the viral titers and pathogenicity in mice.This study indicated that the PB2 protein of H9N2 subtype avian influenza virus exhibited characteristics of mammalian adaptive evolution,which could enhance viral pathogenicity in mice,posing a risk of cross-species infection.