Highly Pathogenic Avian Influenza H5N1 Viruses Elicit an Attenuated Type I Interferon Response in Polarized Human Bronchial Epithelial Cells

Abstract

The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3-and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-␤ compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

“…2B) 3C) and in mouse lungs (Fig. 4) (25,30). The concept that efficient viral growth in the upper respiratory tract of humans can facilitate virus excretion by coughing and sneezing, prompted us to evaluate the growth and release of the virus in polarized oropharyngeal (Detroit 562) cells.…”

“…Cultures were set up with or without TPCK-treated trypsin (1 g/ml; Sigma). Apical and basolateral supernatants were collected at the indicated times, and virus content was determined in a standard plaque assay (25). The values shown represent the mean virus titer of supernatants from three replicate infected cultures.…”

“…To determine whether the 1918 virus genes responsible for the high virulence in vivo also contribute to optimal virus replication in human airway cells, we assessed the growth and release of virus in primary human bronchial epithelial (NHBE) cells (25) and in Detroit 562 epithelial cells derived from a human pharyngeal carcinoma (26). Culture medium from virus-inoculated NHBE cells was collected from the apical and basolateral chambers of the cell monolayers at different times after inoculation and examined for virus production in the presence and absence of trypsin.…”

“…Primary human bronchial epithelial (NHBE) cells (Cambrex Bio Science) (25) and Detroit 562 epithelial cells (American Type Culture Collection) (26), were grown in MEM as described in ref. 25.…”

“…Primary human bronchial epithelial (NHBE) cells (Cambrex Bio Science) (25) and Detroit 562 epithelial cells (American Type Culture Collection) (26), were grown in MEM as described in ref. 25. Briefly, cells (5 ϫ 10 5 ) were seeded onto Corning 24-mm diameter semipermeable membrane inserts with 0.4-m pore size and cultured for 1 week to achieve a stable transepithelial resistance of Ͼ1,000 ⍀⅐cm 2 .…”