Anti-H7N9 avian influenza A virus activity of interferon in pseudostratified human airway epithelium cell cultures

Abstract

Background: Since H7N9 influenza A virus (H7N9) was first reported in 2013, five waves of outbreaks have occurred, posing a huge threat to human health. In preparation for a potential H7N9 epidemic, it is essential to evaluate the efficacy of anti-H7N9 drugs with an appropriate model.

Methods: Well-differentiated pseudostratified human airway epithelium (HAE) cells were grown at the air-liquid interface, and the H7N9 cell tropism and cytopathic effect were detected by immunostaining and hematoxylin-eosin (HE) staining. The H7N9 replication kinetics and anti-H7N9 effect of recombinant human α2b (rhIFN-α2b) and rhIFN-λ1 were compared with different cell lines. The H7N9 viral load and interferon-stimulated gene (ISG) expression were quantified by real-time PCR assays.

Results: H7N9 could infect both ciliated and non-ciliated cells within the three-dimensional (3D) HAE cell culture, which reduced the number of cilia and damaged the airways. The H7N9 replication kinetics differed between traditional cells and 3D HAE cells. Interferon had antiviral activity against H7N9 and alleviated epithelial cell lesions; the antiviral activity of rhIFN-α2b was slightly better than that of rhIFN-λ1. In normal cells, rhIFN-α2b induced a greater amount of ISG expression (MX1, OAS1, IFITM3, and ISG15) compared with rhIFN-λ1, but in 3D HAE cells, this trend was reversed.

Conclusions: Both rhIFN-α2b and rhIFN-λ1 had antiviral activity against H7N9, and this protection was related to the induction of ISGs. The 3D cell culture model is suitable for evaluating interferon antiviral activity because it can demonstrate realistic in vivo-like effects.

Keywords: Avian influenza A virus; H7N9; Human airway epithelium; Interferon; Interferon-stimulated genes.

Fig. 1

Immunofluorescence of 3D HAE cells infected with H7N9. At 24 h post-inoculation with H7N9, the 3D HAE cells were fixed, followed by double immunostaining with anti-influenza A M2 polyclonal antibody (green) and mouse monoclonal anti-β tubulin antibody (red, a) or mouse monoclonal anti-ZO-1 antibody (red, b). Confocal images were taken with a magnification of 100×. Nuclei were stained with DAPI

Fig. 2

Replication characteristics of H7N9 on A549 cells and 3D HAE cells. A549 cells (a) and 3D HAE cells (b) were each incubated with 0.25 μg/ml rhIFN-λ1 or 60 IU/ml rhIFN-α2b for 20 h and then infected with 1 TCID₅₀ of H7N9 virus. Viral RNA was extracted once per day for 10 days. The H7N9 HA gene was quantitatively detected by real-time PCR

Fig. 3

Anti-H7N9 bioactivity of different rhIFN types. A549 cells (a), 2D HAE cells (b), and 3D HAE cells (c) were each incubated with successive 4⁰- to 4⁹-fold dilutions of 4 μg/ml rhIFN-λ1 or 1000 IU/ml rhIFN-α2b for 20 h and then challenged with 100 TCID₅₀ of H7N9. At 72 h post-inoculation, anti-H7N9 bioactivity of different rhIFNs were compared as described above

Fig. 4

Cytopathic effect of rhIFN on 3D HAE cells. After being incubated for 24 h with rhIFN-λ1 or rhIFN-α2b, the 3D HAE cells were infected with 100 TCID₅₀ of H7N9 for 24 h. The cells were then fixed and stained with HE, and images were taken with a microscope at a magnification of 40×

Fig. 5

Effect of rhIFN treatment on ISG induction in different cells. After treatment of A549 cells, 2D HAE cells, and 3D HAE cells with rhIFN-α2b or rhIFN-λ1, the total RNA was extracted and reverse transcribed. The relative expression levels of ISG15, MX1, OAS1, and IFITM3 were measured using Taqman real-time PCR assays and quantified by the 2^−ΔΔCT method. *P < 0.05; **P < 0.01