Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection - PubMed (original) (raw)

Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection

Hui Zeng et al. J Virol. 2012 Jan.

Abstract

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

PubMed Disclaimer

Figures

Fig 1

Fig 1

Detection and quantification of α-2,3- and α-2,6-linked sialic acid (SA) receptors on the cell surface of pulmonary microvascular endothelial cells. (A) Fluorescence lectin staining. Primary human lung blood microvascular endothelial cells (HMVEC-LBI) and human lung microvascular endothelial cells (HULEC) were grown on collagen-coated chamber slides overnight. The cells were incubated with no lectin (control), biotinylated SNA, MAA I, or MAA II, followed by FITC-conjugated avidin D (green) and DAPI (blue) for nuclei. (B) Trypsinized endothelial cells were incubated with FITC-conjugated SNA or MAA (5 μg/ml) and analyzed using flow cytometry. Fluorescent intensities for the α-2,6-SA configuration (SNA, green) and the α-2,3-SA configuration (MAA, blue) were compared to that for unstained cells (red). (C) Virus binding assay. HMVEC-LBI cells grown on collagen-coated chamber slides were incubated with FITC-labeled influenza virus at comparable HAU titers, followed by anti-FITC–horseradish peroxidase (HRP) antibody and AEC detection. The images were taken using the same exposure. (D) Fluorescent lectin staining. Primary rat pulmonary microvascular endothelial cells (PMVEC) and primary rat pulmonary arterial endothelial cells (PAEC) were stained green.

Fig 2

Fig 2

Replication of influenza viruses in human pulmonary microvascular endothelial cells. Endothelial cells grown on six-well plates were infected with influenza virus at an MOI of 0.01. Culture supernatants were collected, and viral titers were determined by a standard plaque assay. Values represent means from three independent experiments plus standard deviations. (A and C) Virus replication in HMVEC-LBI. Ten percent normal chicken egg allantoic fluid was added to the medium during virus infection. (B) Virus replication in HULEC. TPCK-trypsin (0.3 μg/ml) was added to the medium during infection of H1 and H3 subtype viruses. (D) Virus replication in HULEC in the presence or absence of TPCK-trypsin. (E) Immunofluorescence detection of nucleoprotein (NP) antigen in infected HMVEC-LBI and HULEC. Cells seeded on chamber slides were infected with virus at an MOI of 1 and fixed for NP detection at 10 and 24 h p.i.

Fig 3

Fig 3

Replication of avian influenza viruses in human pulmonary microvascular endothelial cells. Endothelial cells grown on six-well plates were infected with influenza virus at an MOI of 0.01. Culture supernatants were collected, and viral titers were determined by a standard plaque assay. Values represent the means from three independent experiments plus standard deviations. (A) Virus replication in HULEC. TPCK-trypsin (0.3 μg/ml) was added to the medium only for cells infected with Dk/NY virus. (B) Virus replication in HMVEC-LBI. Ten percent normal chicken egg allantoic fluid was added to the medium during virus infection. *, H5N1 virus replicated to a significantly higher titer than H7 subtype viruses (P < 0.05) at 48 h and 72 h p.i.

Fig 4

Fig 4

HPAI H5N1 viruses preferentially enter from the apical side of polarized human pulmonary microvascular endothelial cells. HULEC monolayers grown on transwells for 1 week were infected with VN/1203 virus apically or basolaterally at an MOI of 0.01. The supernatant from both apical and basolateral compartments was collected for virus titration using standard plaque assay. Values represent the means from three independent experiments plus standard deviation. aa, apical infection and apical release; ab, apical infection and basolateral release; ba, basolateral infection and apical release; bb, basolateral infection and basolateral release.

Fig 5

Fig 5

Contribution of the HA multiple basic amino acid cleavage site to virus replication in human endothelial cells. Cells were infected with virus at an MOI of 0.01, and supernatants were collected for virus titration by plaque assay. (A) Immunofluorescence detection of nucleoprotein (NP) antigen in infected HMVEC-LBI. Cells seeded on chamber slides were infected with virus at an MOI of 1 and fixed for NP detection at 8 h p.i. (B) Virus replication in polarized bronchial epithelial cells (Calu-3). (C) Virus replication in HMVEC-LBI. Ten percent normal chicken egg allantoic fluid was added to the medium. For real-time PCR analysis, cells were infected with each virus at an MOI of 1 and RNA was collected at 24 h p.i. (D) Relative M1 gene levels in infected cells determined by real-time PCR. (E) Relative fold change of gene expression of type I interferon genes in infected cells.

Fig 6

Fig 6

Evaluation of cytokine production in virus-infected endothelial cells. HULEC were infected with virus at an MOI of 1, and supernatants were collected at 24 h p.i. for Bio-Plex assay. Values represent the means from three independent experiments plus standard deviations.

Fig 7

Fig 7

Examination of endothelial cell proliferation/viability during influenza virus infection. HMVEC-LBI were seeded onto 96-well plates, inoculated with virus at an MOI of 5, 1, or 0.2, and examined at 44 h p.i. using WST-1 reagent. *, infection with VN/1203 virus resulted in significantly lower levels of cell variability compared to other tested viruses at all virus dilutions (P < 0.05). Values represent the means from three independent experiments plus standard deviations.

Similar articles

Cited by

References

    1. Abdel-Ghafar AN, et al. 2008. Update on avian influenza A (H5N1) virus infection in humans. N. Engl. J. Med. 358:261–273 - PubMed
    1. Alom-Ruiz SP, Anilkumar N, Shah AM. 2008. Reactive oxygen species and endothelial activation. Antioxid. Redox Signal. 10:1089–1100 - PubMed
    1. Azoulay E, et al. 2002. Deterioration of previous acute lung injury during neutropenia recovery. Crit. Care Med. 30:781–786 - PubMed
    1. Baskin CR, et al. 2009. Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus. Proc. Natl. Acad. Sci. U. S. A. 106:3455–3460 - PMC - PubMed
    1. Belser JA, et al. 2008. Contemporary North American influenza H7 viruses possess human receptor specificity: implications for virus transmissibility. Proc. Natl. Acad. Sci. U. S. A. 105:7558–7563 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources