Kinetic Characterization of PB1-F2-Mediated Immunopathology during Highly Pathogenic Avian H5N1 Influenza Virus Infection

The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.甲型流感病毒编码的PB1-F2蛋白可增强病毒毒力，该功能依赖于其序列多态性。然而，部分H1N1亚型病毒的PB1-F2蛋白已被证实可加剧气道内炎症反应，但PB1-F2对哺乳动物体内高致病性禽流感病毒（HPAIV）毒力的贡献，目前仍鲜有研究阐明。本研究采用一株从鸭体内分离的H5N1亚型HPAIV毒株及其PB1-F2基因敲除突变株，在致死性肺炎小鼠模型中解析了PB1-F2相关宿主应答的动态变化过程。两种毒株感染小鼠的平均死亡时间均为10天，这使得我们能够在多个时间点对感染肺部开展全转录组分析及详细的组织病理学检测。感染后第2天（pi），虽未观察到组织病理学损伤，但PB1-F2的表达可显著抑制巨噬细胞活化相关的细胞通路，并呈现出提示其会促进上皮屏障损伤的转录组特征。感染后第4天，与PB1-F2表达相关的基因谱显示自然杀伤（NK）细胞活性出现异常。感染后第8天，PB1-F2的表达与趋化因子及细胞因子编码基因的转录上调显著相关——这些趋化因子和细胞因子参与粒细胞的招募；同时还与中性粒细胞表达的多种酶编码基因的表达上调相关。小鼠肺部的组织病理学分析完全验证了上述转录组数据：在PB1-F2表达的情况下，肺部炎症损伤更为严重，中性粒细胞招募也更为显著，这为本研究所得出的结论提供了功能层面的佐证。综上，本研究表明，H5N1亚型HPAIV的PB1-F2蛋白对宿主转录组的调控方式与H1N1亚型PB1-F2存在显著差异：H5N1 PB1-F2首先会延迟初始免疫应答，但在感染后期会加剧肺部炎症反应。