Development of an influenza A virus and Aspergillus fumigatus coinfection model

摘要 入住ICU的甲型流感病毒(IAV)感染者面临严重继发感染 并发症。越来越有力的证据表明，曲霉继发性真菌感染 烟雾菌(Af)是严重IAV感染的ICU患者的重要危险因素。总的来说， ICU中约20%的重症IAV患者可能发生继发性房颤感染。目前， 在理解这些真菌继发感染是如何发展的以及为什么会发展的问题上存在着严重的差距。 在IAV患者中。这个R21应用程序旨在通过生成一个健壮的小动物IAV-Af来填补这一空白 可用于剖析鸡传染性支气管炎病毒感染分子机制的共感染模型(S) 增加对Af挑战的易感性。我们最初的初步实验表明，IAV感染4 Af攻击前几天会导致显著的真菌生长和组织侵袭，这在 幼稚的小鼠挑战Af。在这个R21提案的SA1中，我们将进一步优化和发展这一点 IAV-Af混合感染模型，以确保其高度重复性并确定IAV血清型的重要性 临床菌株对混合感染的易感性存在异质性。在SA2中，我们将检验假设 IAV感染通过两种创新技术损害肺白细胞抗真菌活性：1) 荧光曲霉报告(FLARE)菌株直接定量检测白细胞的抗真菌活性 单细胞水平的群体和2)单细胞RNA测序(scRNA-Seq)以监测 肺白细胞群体中无偏倚的转录变化。这些数据将 首次深入了解IAV感染后Af易感性的驱动机制。总体而言，这 研究填补了一个关键空白，为该领域提供了一种独特的、急需的IAV-Af混合感染 模型，并为未来的重点、机械化的发展提供了一个独特的、健壮的数据集 R01应用程序。

ABSTRACT Influenza A virus (IAV) infected patients admitted to the ICU face severe secondary infection complications. There is increasingly strong evidence that secondary fungal infections with Aspergillus fumigatus (Af) present a significant risk for ICU patients with severe IAV infection. Overall, approximately 20% of severe IAV patients in the ICU may develop secondary Af infection. Currently, there is a critical gap in understanding how and why these fungal secondary infections develop in IAV patients. This R21 application aims to fill this gap by generating a robust small animal IAV-Af coinfection model that can be used to dissect the molecular mechanism(s) by which IAV infection increases susceptibility to Af challenge. Our initial pilot experiments demonstrate that IAV infection four days prior to Af challenge results in significant fungal growth and tissue invasion, which is absent in naïve mice challenged with Af. In SA1 of this R21 proposal we will further optimize and develop this IAV-Af coinfection model to ensure it is highly reproducible and define the importance of IAV serotype and Af clinical strain heterogeneity in susceptibility to coinfection. In SA2, we will test the hypothesis that IAV infection impairs pulmonary leukocyte anti-fungal activity using two innovative techniques: 1) Fluorescent Aspergillus reporter (FLARE) strains to directly quantify anti-fungal activity of leukocyte populations at a cell single level and 2) Single cell RNA sequencing (scRNA-Seq) to monitor transcriptional changes in pulmonary leukocyte populations in an unbiased manner. These data will provide the first insights into mechanisms driving Af susceptibility following IAV infection. Overall, this research fills a critical gap by providing the field with a unique and highly needed IAV-Af coinfection model and provides a unique and robust dataset for the development of future focused, mechanistic R01 applications.