Department of Microbiology, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, NY 10029, USA.
Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun 7.
Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b(+)CD11c(-), CD11b(-)CD11c(+), and CD11b(+)CD11c(+), as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.
甲型流感病毒因其对人类和动物健康的重大影响而受到广泛研究。然而,流感病毒感染的动力学以及体内感染的细胞类型仍知之甚少。这些特征难以确定,部分原因是没有能够表达易于追踪的报告基因的高效复制型病毒。在这里,我们报告了一种携带 GFP 报告基因的重组流感病毒(NS1-GFP 病毒)的产生。尽管与野生型病毒相比,该 NS1-GFP 病毒的毒性减弱,但它在小鼠肺部中高效复制并表现出致病性。使用全器官成像和流式细胞术,我们追踪了流感病毒在小鼠体内感染进展的动力学。对小鼠肺部的成像显示,感染首先从靠近大导气道的呼吸道区域开始,然后扩散到肺部的更深部位。除了上皮细胞,我们还发现了 GFP 阳性的抗原呈递细胞,如 CD11b(+)CD11c(-)、CD11b(-)CD11c(+)和 CD11b(+)CD11c(+),在鼻内感染后 24 小时即可检测到。此外,相当比例的 NK 和 B 细胞呈 GFP 阳性,表明这些细胞存在活跃的感染。接下来,我们测试了流感病毒抑制剂奥司他韦和金刚烷胺对体内感染进展动力学的影响。奥司他韦治疗显著降低了所有细胞类型的流感感染,而令人惊讶的是,金刚烷胺治疗更有效地阻断了 B 和 NK 细胞的感染。我们的结果表明,在病毒感染期间存在大量携带流感病毒抗原的免疫细胞,并且在使用抗病毒药物治疗时存在细胞类型特异性效应,为流感病毒领域的研究开辟了新的途径。
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