重组rSD01H9N2禽流感病毒感染A549细胞及其诱导的免疫反应

Recombinant rSD01 H9N2 avian influenza virus infects A549 cells and induces immune response

为了解禽流感病毒(AIV)与其他病毒重组带来的风险,本试验利用反向遗传技术,以H9N2A/Chicken/Shandong/01/2008(SD01)株为骨架,将其PA基因替换为H1N1A/swine/Shandong/07/2011(SD07)株的PA基因,构建了1株重组病毒rSD01-PA。以A549细胞为模型,感染H9N2AIV SD01及其重组株rSD01-PA。通过间接免疫荧光(IFA)和荧光定量PCR(qRT-PCR)的方法,检测H9N2AIV及其重组株在A549细胞上的复制情况,以及4种模式识别受体(TLR-3、TLR-7、RIG-I和MDA-5)和4种细胞因子(IFN-β、IL-6、MX和OAS)。结果显示:SD01和重组株rSD01-PA在A549细胞上均能有效复制,且重组株rSD01-PA复制能力较强。同时2株毒均能引起4种模式识别受体和4种细胞因子显著上调。在病毒感染的过程中,rSD01-PA感染A549细胞引起的炎性细胞因子IFN-β、IL-6和MX的表达水平较SD01更高。本试验结果将有助于进一步认识H9N2亚型AIV与其他病毒重组带来的风险以及病毒感染后宿主的防御机制及感染反应。

In order to know about the risk of recombination caused by avian influenza virus(AIV)and other viruses,by reverse genetics,a reassortant rSD01-PA was derived from the H9N2 subtype AIV A/Chicken/Shandong/01/2008(SD01)by introducing the PAgene from the pandemic influenza A H1N1 virus A/swine/Shandong/07/2011(SD07).A549 cells were set as a model,which infected by H9N2 subtype AIV SD01 and the reassortant rSD01-PA.Through replication of the SD01 and the rSD01-PA on A549 cells,4kinds of pattern recognition receptors(TLR-3,TLR-7,RIG-I and MDA-5)and 4kinds of cytokines(IFN-β,IL-6,MX and OAS)were detected by indirect immunofluorescence assay(IFA)and quantitative real-time polymerase chain reaction(qRT-PCR).Results show that the SD01 and the rSD01-PA can be replicated effectively on A549 cells,and the replication ability of the rSD01-PA was stronger.At the same time,they could all caused 4kinds of pattern recognition receptors and 4kinds of cytokines to upregulate significantly.During the viral infection,the expression levels of inflammatory cytokines IFN-β,IL-6and MX on A549 cells infected by the rSD01-PA were higher than those of the SD01.The results help to further understand the risk of recombination,as well as the defense mechanism and the reaction of the host after the virus infection.