Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence.
Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence.
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DOI：
10.1186/1743-422x-8-13
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发表时间：
2011-01-12
期刊：
影响因子：
4.8
通讯作者：
Brown EG
中科院分区：
文献类型：
--
作者：
Dankar SK;Wang S;Ping J;Forbes NE;Keleta L;Li Y;Brown EG
为了理解病毒在新宿主中变得有毒所需的进化步骤，使人甲型流感病毒（IAV）A/Hong Kong/1/68（H3 N2）（HK-wt）适应于在小鼠中增加毒力。在NS 1基因中选择的11个突变中，先前在高毒力人H5 N1分离株A/HK/156/97中检测到两个突变F103 L和M106 I，表明这些突变在小鼠和人类中的毒力中的作用。为了确定这些突变的选择性优势，使用反向遗传学将含有每种NS 1小鼠适应性突变的病毒拯救为在A/PR/8/34遗传骨架上具有HK-wt NS 1基因的病毒。F103 L和M106 I NS 1突变均显著增强体外（小鼠和犬细胞）和体内（BALB/c小鼠肺）生长，并增强小鼠毒力。只有M106 I NS 1突变增强了人类细胞的生长。此外，这些NS 1突变增强了MDCK细胞中的早期病毒蛋白质合成，并显示出相对于rPR 8-HK-NS-wt NS 1，在小鼠干扰素β（IFN-β）预处理的小鼠细胞中复制的能力增加。双突变体rPR 8-HK-NS-F103 L + M106 I由于小鼠细胞中IFN-β诱导增加而在感染后期表现出生长衰减。然后，我们产生了具有A/HK/156/97 NS基因的rPR 8病毒，该基因具有103 L +106 I，然后拯救了L103 F + I106 M突变体。103 L +106 I突变在BALB/c小鼠中使毒力增加>10倍。我们还将具有103 L +106 I的禽A/Ck/Beijing/1/95 NS 1基因（A/HK/156/97 NS 1基因的来源谱系）插入到A/WSN/33骨架上，然后产生L103 F + I106 M突变体。含病毒的H5 N1和H9 N2 NS均未导致IFN-β诱导增加。具有103 L和106 I的rWSN-A/Ck/Beijing/1/95-NS 1基因表现出100倍的生长增强和>10倍的毒力增强，这与相对于相应的L103 F和I106 M突变体对肺泡和细支气管组织的增加的嗜性相关。在小鼠模型中，F103 L和M106 I NS 1突变是人类和禽类NS 1基因中生长和毒力的适应性遗传决定因素。
To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans. To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant. The F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.