新现H3N2亚型SIV感染人肺腺癌细胞病毒基因变化及跨种属感染相关miRNA研究

We have isolated and identified several viral strains which are re-assorted with H3N2 swine influenza virus (swine influenza virus, SIV) and 2009 H1N1 pandemic influenza virus (pdm/09) in GX during 2010-2014. It is found that these viruses have infected humans. But the molecular mechanism is not clear about the genetic changes and microRNA study associated with cross-species infection for the emergent H3N2 SIV of GX human infected lung cancer A549 cells. So we would like select one presented isolated strain which is named SW2783 for the following study: In the first part of experiment, SW2783 is re-infected human lung cancer A549 cell line for 10 cycles of infection and genome sequence of progeny SW2783 virus will be performed. When a meaningful genetic change come to our observation, especially on the hemagglutinin (HA), then we reconstruct re-assortment virus rH3N2 SIV with a single residue substitution at this site. Then we reconstruct re-assortment virus rH3N2 SIV with a single residue substitution at this site. After then, the in vitro infection of freshly-excised human nasal, tracheal, bronchial and lung tissues by rH3N2 SIV will be researched to explore their tissue tropism ability; For the second part of experiment, the different expression microRNAs will be detected by the RNA sequence after human lung cancer A549 cells infected by SW2783 virus and controlled virus. The significant microRNAs associated with cross-species infection for the emergent H3N2 SIV of GX infected human lung cancer A549 cells will be analyzed and their functions will be tested. The cross-action for the proteins between host cells and SW2783 virus will be studied and the viral targeted genes will be determined. This study will provide some clues about the genes and microRNAs associated with cross-species infection for the emergent H3N2 SIV of GX after infecting human cells.

2010-2014年我们得到若干株由新甲型 H1N1 流感病毒与H3N2 亚型猪流感病毒重组产生的新现H3N2 SIV病毒，且有证据表明该病毒已感染人，但尚未清楚该病毒跨种属感染人分子机制。为此，我们拟选择代表毒株SW2783进行如下研究：首先，SW2783连续感染人肺腺癌A549细胞，分析其感染人细胞后病毒基因变化，然后对突变毒株突变氨基酸进行替换，拯救重组病毒，分别与胎儿呼吸系统各段组织进行病毒感染结合实验，分析病毒对呼吸道组织亲嗜力，了解该病毒感染人后出现的病毒基因改变；其次，用RNA深度测序对SW2783病毒和对照毒株病毒感染人肺腺癌A549细胞的miRNA进行差异表达分析、功能验证， 以及差异表达miRNA与H3N2亚型SIV的病毒蛋白互作检测和病毒靶基因确定，以探讨该病毒感染人细胞后的宿主跨种属感染人相关miRNA。研究为揭示新现H3N2 SIV跨种属感染人分子机制提供线索。

本项目关注miRNA在流感病毒感染过程中发挥的作用，主要侧重在具有跨种属感染潜力的新现SIV感染A549细胞过程中是否存在特殊miRNA这一问题上，我们利用高通量深度测序和生物信息学大量数据分析，绘制新现SIV感染过程中miRNA-mRNA-蛋白质互作图谱，寻找特异性差异表达的miRNA，探讨其对病毒复制的影响，进一步研究微小RNA在开发抗病毒药物、研制疫苗和筛选新的生物标记物的潜力。得到如下结论：1. 新现SW2783在体内外实验中均提示其具有跨种属感染的特征。2. 通过miRNA-mRNA整合网络分析，筛选出3个非编码miRNA ：hsa-miR-140-5p, hsa-miR-30a-3p和hsa-miR-582-5p，它们在SW2783感染过程中呈现特异性的差异表达，推测可能与跨种属感染有密切关系。3. 通过miRNA-mRNA-蛋白质整合网络分析，筛选出非编码miRNA：hsa-miR-140-5p与3个潜在的靶蛋白（ERVFRD-1，PPIA和IFIT2）可能存在调控关系，并有可能对有跨种属感染的毒株在A549细胞中的感染能力有影响。4. 通过功能实验证实hsa-miR-140-5p能够对SW2783感染A549细胞有特殊调控作用，表现在其能影响病毒的复制，并影响其靶向的ERVFRD-1，PPIA和IFIT2的表达。综上，在SW2783感染A549细胞过程中，非编码miRNA—hsa-miR-140-5p发挥了特殊的调控病毒复制作用，可能作为筛选跨种属感染的新型流感病毒的潜在生物标志物，或者治疗的新型药物靶点。