Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread

Abstract

The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.

Fig 1

Generation of an influenza reporter virus encoding a PA-NLuc fusion. (A) Schematic of the PA-NLuc gene fusions encoding the polyprotein containing PA, the self-cleaving 2A peptide where indicated, and NLuc. Repeated regions of the PA ORF that contain packaging signals and are repeated downstream of NLuc are depicted as dark gray boxes. Silent mutations were introduced into the terminal 47 nt of the PA coding sequence in the PA-SWAP-2A-NLuc50 (PASTN) gene. (B) Polymerase activity assay of PA fusions with or without the 2A peptide and with packaging sequence repeats of increasing length. Polymerase activity assays were performed with human 293T cells by transfecting vectors expressing PB1, PB2, the indicated PA proteins, NP, and a vNA-based firefly luciferase reporter. PA proteins were detected by Western blotting. Data are presented as means ± standard deviations (sd) (n = 3).

Fig 2

PA-2A-NLuc permits exquisitely sensitive detection of an influenza reporter virus. (A) Nano-Glo viral titer assay performed with defined amounts of input virus. Where indicated, cells were pretreated with 100 μg/ml ribavirin, which was then present throughout the assay. A trend line (gray dashed line) was fitted to untreated samples. *, P < 0.005; n = 4 ± sd. (B) Multicycle replication in human Calu-3 lung cells infected at MOI = 0.001. Viral titers were determined by both a plaque assay and a Nano-Glo viral titer assay (n = 3 ± sd). The limit of detection for luciferase activity is indicated as a black dashed line in panels A and B. To demonstrate the relationship between the two titer measurements, RLU was plotted as a function of PFU (inset). (C) Multicycle replication kinetics of native and PASTN viruses in MDBK cells infected at MOI = 0.01. Viral titers were determined by a plaque assay. Titers of WSN and PASTN at 24 and 39 h were significantly different (*, P < 0.05; n = 3 ± sd). (D) The PASTN gene is stable in vivo and through multiple passages in culture. RT-PCR was performed on RNA recovered from viral stocks (passage 0 [P0]), mouse lung homogenate from 5 dpi, or virus following serial passage in MDBK or Calu-3 cells. The PCR product was analyzed by agarose gel electrophoresis. Expected product sizes for native PA and the full-length reporter gene are indicated.

Fig 3

In vivo imaging of an influenza reporter virus with near-native replication properties. Noninvasive imaging detects robust bioluminescence in mice infected with PASTN. (A) Mice were infected with 10⁴ PFU of PASTN virus and analyzed for weight loss and bioluminescence. Representative data from serial imaging of one mouse are shown. (B) In a separate experiment, weight loss was measured for cohorts of mice infected with 10⁴ PFU of either WSN or PASTN virus (n = 3 ± sd). (C) Cohorts of mice were infected as described for panel B and sacrificed at the indicated times. Viral titers in lung homogenates were determined by TCID₅₀ and a Nano-Glo titer assay (n = 3 ± sd).

Fig 4

The NLuc influenza reporter virus recapitulates viral attenuation due to species-specific restriction of polymerase function. Mice were infected with 10⁴ PFU of PATN virus encoding either WT PB2 or PB2 with the avian-signature K627E mutation. Flux was determined for mice imaged individually (2.5 dpi) or concurrently (4 dpi).

Fig 5

The PASTN reporter virus retains near-native pathogenicity in vivo. (A) BALB/c mice were infected intranasally with 10², 10³, or 10⁴ PFU of PASTN virus, and body weights were measured for 14 days (n = 4 + sd). (B) Survival rates of mice infected as described for panel A. (C) The relationship between bioluminescent signal and viral dose was examined by longitudinal observation of mice infected with increasing doses of PASTN virus. (D) Serial observations over the course of a sublethal infection of a single mouse from the experiment described for panel C with 10² PFU of PASTN virus permitted real-time observation of viral clearance. The same scale was used for all mouse images.