Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus

Abstract

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.

Fig. 1.

Generation of recombinant influenza virus carrying a GFP reporter. (A) Schematic representation of NS segment of WT PR8 virus and NS1-GFP virus. Splice acceptor site in NS was mutated to prevent mRNA splicing (SD-splice donor site, SA- splice acceptor site). Common regions present in both NS1 (light blue) and NEP (dark blue) are shown in red. NS1was fused to GFP (green) via a GSGG linker, followed by PTV-1 2A autproteolytic cleavage site (yellow) and NEP ORF (red-blue). (B) A549 cells were infected with recombinant PR8 virus carrying NS1-GFP. At 10 hpi, cells were fixed and stained for NP. NP staining is shown in red and NS1-GFP is shown in green. (C) Fluorescent micrographs of NS1-GFP virus plaques taken at 20× magnification.

Fig. 2.

In vivo characterization of NS1-GFP virus. (A and B) Comparison of survival of PR8 and NS1-GFP virus–infected mice. BALB/c mice were intranasally inoculated with indicated doses of PR8 or NS1-GFP virus. Survival was monitored daily. (C) Viral titers in lungs of mice infected with WT PR8 virus and with NS1-GFP viruses. (D) Mice were intranasally inoculated with either PR8 or NS1-GFP virus at indicated doses. Lungs were excised on day 4 postinfection, and fluorescence from infected lungs was imaged using an IVIS-200 imaging system (Xenogen). (E) Fluorescent micrographs of mice lung cryosections (10× magnification).

Fig. 3.

Dynamics of influenza virus infection in lungs. (A) Kinetics of epithelial cell infection. BALB/c mice were intranasally inoculated with NS1-GFP virus at indicated doses, and lung homogenates were analyzed for GFP expression in nonhematopoietic cells (CD45⁻) using a BD LSR II flow cytometer. (B) Comparison of kinetics of hematopoietic and nonhematopoietic cell infection in murine lungs. BALB/c mice were intranasally inoculated with 10⁶ pfu NS1-GFP virus and analyzed for GFP expression in cells types differentially expressing CD11b and CD11c. Each data point represents the average from at least three mice.

Fig. 4.

Oseltamivir and amantadine treatment significantly reduces NS1-GFP virus infection. Mice infected with NS1-GFP virus either were left untreated or were treated with oseltamivir (50 mg/kg) or amantadine (40 mg/kg), starting 1 h postinfection. Kinetics of infection progression in different cell types were analyzed using a BD LSR II flow cytometer. (A–G) Kinetics of GFP expression in different cell types as indicated in treated and untreated groups.

Fig. 5.

Oseltamivir treatment restricts influenza virus infection to localized areas. Ex vivo imaging of mice lungs on day 2 (A) and day 4 (B) postinfection. BALB/c mice infected with NS1-GFP virus (10⁶ pfu) either were left untreated or were treated daily once with oseltamivir (50 mg/kg). Lungs of mice were excised at indicated time and imaged using the IVIS-200 system (Xenogen).