Development of an influenza A virus and Aspergillus fumigatus coinfection model

摘要 A型流感病毒（IAV）感染患者进入ICU后面临严重的继发感染 并发症越来越多的有力证据表明，继发性真菌感染曲霉菌 烟曲霉（Af）对重症IAV感染的ICU患者具有显著风险。总体而言， ICU中约20%的重症IAV患者可能发生继发性Af感染。目前， 在理解这些真菌继发感染如何以及为什么发生方面存在着严重的差距 在IAV患者中。这项R21应用旨在通过产生强大的小动物IAV-Af来填补这一空白 共感染模型，可用于剖析IAV感染的分子机制 增加对Af攻击的敏感性。我们初步的试验表明，IAV感染4 在Af攻击前30天导致显著的真菌生长和组织侵入，这在Af攻击中是不存在的。 在本R21提案的SA 1中，我们将进一步优化和开发这种 IAV-Af共感染模型，以确保其具有高度可重复性，并确定IAV血清型的重要性 和Af临床菌株对合并感染的易感性的异质性。在SA 2中，我们将检验假设 IAV感染损害肺白细胞抗真菌活性，使用两种创新技术：1） 荧光曲霉报告菌株（FLARE），用于直接定量白细胞的抗真菌活性 2）单细胞RNA测序（scRNA-Seq），以监测 以无偏的方式检测肺白细胞群体中的转录变化。这些数据将 提供了第一个深入了解驱动Af易感性IAV感染后的机制。总的来说，这 研究填补了一个关键的空白，为该领域提供了一个独特的和高度需要的IAV-Af合并感染 模型，并提供了一个独特的和强大的数据集的发展，未来的重点，机械 R 01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.