Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1

Abstract

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

Figure 1

Cell death in avian embryo fibroblasts following influenza A infection. (a) Duck and (b) chicken embryo fibroblasts infected with classical swine H1N1 virus for 48 h. (c) Uninfected duck and (e) chicken cells are shown for comparison. Immunostaining for viral nucleoprotein antigen in (d) duck and (f) chicken cells 6 h following viral infection at an MOI of 1.0. Similar results were obtained following infection with LPAI H2N3 or with HPAI H5N1 50-92 viruses.

Figure 2

Duck lung cells show lower metabolic activity compared with chicken lung cells following influenza A infection. Measurement of cell viability in duck and chicken lung cells, using an MTT assay, 24 h post infection, with a range of MOI, using (a) LPAI H2N3 or (b) classical swine H1N1 virus. Data points are the mean of quadruplicate wells with error bars showing s.d. There is a significant difference between species (P<0.01; two-way analysis of variance).

Figure 3

Duck cells show higher levels of hypodiploid cells than chicken lung cells following influenza A infection. Measurement of cellular levels of DNA fragmentation following influenza A virus infection by propidium iodide staining of ethanol-fixed cells, followed by flow cytometry. Duck lung cells (black bars) and chicken lung cells (white bars) were infected for 24 or 48 h with low pathogenic avian (a) H2N3, (b) classical swine H1N1 or for 20 h with (c) highly pathogenic H5N1 50-92 or (d) H5N1 tyTy05. Analysis of data by three-way analysis of variance for interspecies differences showed a significant difference between species (P<0.01) in the proportion of hypodiploid cells following infection with low pathogenic avian H2N3 (a), classical swine H1N1 (b) or highly pathogenic H5N1 50-92 (c) but not H5N1 tyTy05 (d; P=0.086). Data shown are the mean of duplicate wells.

Figure 4

Caspase 3/7 induction is greater in duck cells compared with chicken cells following influenza A infection. Chicken (white bars) and duck embryo fibroblasts (black bars) were infected with avian H2N3 or classical swine H1N1 viruses for 18 or 40 h at an MOI of either 1.0 or 0.1. Chemical treatment of cells with staurosporin (4 μ) was used as a positive control. Levels of caspase 3/7 induction were quantified using Caspase-Glo 3/7 Assay (Promega). Data points are the mean of triplicate wells with error bars showing s.d. Analysis of data by three-way analysis of variance showed a significant difference between species (P<0.001) following influenza infection with either virus at both MOI; no significant difference was observed between species following staurosporin treatment (P=0.074). RLU/s, relative light units per second.

Figure 5

Replication of influenza A in duck and chicken cell cultures. Chicken cells (white bars) and duck cells (black bars) were infected with a range of viruses for 20–48 h and virus production measured by real-time PCR and titration of virus in culture supernatants. (a) Duck and chicken lung cells infected with LPAI H2N3 at an MOI of 1.0 for 24 h and virus output measured by detection of viral M gene RNA in supernatant by quantitative reverse transcription PCR. (b) Duck and chicken embryo cells infected with HPAI H5N1 50-92 or H5N1 tyTy05 for 20 h at an MOI of 1.0 and virus production quantified by viral M gene RNA PCR. Significant difference in M gene copy number between species following H5N1 50-92 (P<0.05) but not H2N3 infection or H5N1 tyTy05 infection. (c, d) Infectious virus in supernatants from duck and chicken lung cells following infection for 24 or 48 h, with (c) LPAI avian H2N3 and (d) classical swine H1N1 measured by titration on Madin-Darby canine kidney cells. (e, f) Levels of infectious virus in supernatants following infection of chicken embryo cells and duck embryo cells at an MOI of 1.0 for 20 or 40 h with (e) HPAI H5N1 50-92 or (f) H5N1 tyTy05. Pretreatment with a pan-caspase inhibitor Q-VD-OPh (10 μ) before HPAI H5N1 infections (Block). Analysis of data by three-way analysis of variance, for interspecies differences in the production of infectious virus, showed significant differences (P<0.001) following influenza infection with H5N1 50-92 or tyTy05. No significant difference on the level of infectious virus production was observed following treatment with Q-VD-OPh (P>0.05). Data show the mean of triplicate wells with error bars showing s.d. FFU, focus-forming units.