Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses

Abstract

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography-computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.

Keywords: influenza virus; multi-modal imaging; multiplicity reactivation; reporter virus.

Figure 1

Characterization of a toolbox of influenza PA-reporter viruses. (A) Schematic of PA-reporter gene fusions encoding PA polyproteins containing the 2A peptide (PA-2A-GFP [PATG] and PA-2A-RFP [PATR]) or PA direct fusions that lack the 2A sequence (PA-SWAP-NLuc [PASN]). Silent mutations introduced into the terminal 47 nt of the PA coding sequence are depicted with diagonal lines and the 50 nt repeat of the PA open reading frame (ORF) that maintains the packaging signal is shown; (B,C) Multicycle replication kinetics of parental WSN and PA-reporter viruses in A549 cells infected at a multiplicity of infection (MOI) = 0.01. Viral titers were determined at the indicated time points by plaque assay. Data are mean of n = 3 ± standard deviation (sd). statistically significant differences between WSN and the reporter viruses are marked (* p < 0.05, Student’s t-test).

Figure 2

Bioluminescent virions provide a rapid and quantitative measure of attachment to cells. PASN packages PA-NLuc fusions to create bioluminescent virions. (A) PA fusion proteins were detected by western blotting of lysates from infected cells (left) or virions purified through a 20% sucrose cushion (right); (B) PASN attachment assay. 10⁶ plaque-forming units (pfu) of PASN per well were incubated with A549 or MDCK cells at 37 °C for the indicated time. Where noted, cells were pre-treated with receptor destroying enzyme (RDE) to remove sialic acids. Following washing to remove unbound virus, attached virions were measured in a 96-well NanoGlo assay. Data are mean of n = 3 ± sd; (C) A highly sensitive attachment assay. A549 or MDCK cells were incubated with dilutions of virus at 4 °C for 45 min to allow binding to go to completion. Following washing to remove unbound virus, attached virions were measured in a 96-well NanoGlo assay and the limit of detection was set to background luminescence in the absence of infection. Data are mean of n = 4 ± sd. Statistically significant differences between cells inoculated with PASN or media alone for each cell line are marked (* p < 0.001, Student’s t-test).

Figure 3

Influenza reporter viruses detect infection, co-infection, “semi-infection” and multiplicity reactivation. Live cell imaging of viral gene expression in infected cells. (A) A549 cells were infected with PATG, PATR, WSN or co-infected with PATG and PATR. Infected cells were detected by fluorescence microscopy 18–19 hours post-infection (hpi) and cells were counter-stained with Hoechst 33342 to detect nuclei. Co-infection is indicated by the arrowheads; (B) A549 cells were infected with an increasing MOI of PATG. Cells were imaged to detect total cells (Hoechst 33342) and those expressing a virally encoded gene (GFP). All of the cells are GFP-positive when only an MOI = 0.4 is used; (C) Multiplicity reactivation rescues viral gene expression. A total of 25,000 MDCK cells were infected in a 96-well format with PASTN at an MOI = 0.005. Where indicated, cells were also simultaneously infected with increasing amounts of WSN. A luciferase activity assay was performed 10 hpi to determine PASTN gene expression for each condition (average relative light units (RLU), Data are mean of n = 4 ± sd). Conditions with a statistically significant increase in gene expression compared to PASTN alone are marked (* p < 0.05, Student’s t-test).

Figure 4

Multi-modal imaging of the effect of antiviral treatment on influenza virus-mediated inflammation. (A) Infected mice were treated with oseltamivir (OSV, 10 mg/kg bid) beginning 2 days post infection (dpi) or mock treated; Serial bioluminescence imaging of the same cohort of animals was used to detect statistically significant differences in viral load over time between mock and OSV treatment (p = 0.0085, 2-way ANOVA); (B) Multi-modal bioluminescence and positron emission tomography–computed tomography (PET/CT) imaging. [18F]-2-deoxy-2-fluoro-d-glucose (¹⁸F-FDG) was used as a probe to measure inflammation in the lungs. Representative data from uninfected and infected mice are shown; (C) Dual quantification of viral load (bioluminescent radiance) and inflammation (%-injected dose(ID)/g) in the lungs of infected or control mice. Three different Hounsfield unit thresholds were used to define lung tissue by CT from which ¹⁸F-FDG was quantified by PET. Data were collected from one control mouse, two infected mice, and two infected mice treated with OSV.