SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

Abstract

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

Fig. 1

Growth kinetics of icSARS-CoV in human airway epithelial cells. To determine viral growth kinetics of icSARS-CoV in HAE cultures over time, icSARS-CoV was inoculated onto the apical surface of HAE cultures and apical washes (A) and basolateral media (B) collections were performed at the indicated times. The washes were serially diluted and titers assayed on Vero E6 cells. Titers are expressed as PFU/mL.

Fig. 2

Ultrastructural localization of SARS-CoV in HAE. Representative transmission electron microscopic photomicrographs of HAE infected with Urbani SARS-CoV. (A) HAE inoculated with vehicle alone demonstrating the typical morphological features of the apical surfaces of ciliated cells with prominent cilia (black arrow heads) and microvilli (arrows). (B–F) HAE inoculated with Urbani SARS-CoV 48 h before fixation and showing the presence of large numbers of virus particles (open arrow heads) in vesicles inside the ciliated cells (B and E), and on the surface of ciliated cells (B–F). To confirm the observed virions were SARS-CoV, immuno-EM was performed using polyclonal mouse antisera against S with secondary antibodies conjugated to 12 nm gold beads (D, open arrow heads indicate virions, arrows indicate colloidal gold). SARS-CoV infection resulted in extrusion and shedding of infected ciliated cells into the airway surface microenvironment (F). Similar observations were seen with HAE infected with icSARS-CoV and SARS-CoV GFP. Black arrowheads, cilia; black arrows, microvilli; open arrowhead, virions; small arrow, immuno-EM colloidal gold.

Fig. 3

hACE2 is the primary receptor for SARS-CoV entry into HAE. To assess effects of pretreatment of HAE with receptor specific antisera on the growth kinetics of SARS-CoV infection, HAE were pretreated with polyclonal or monoclonal antisera directed against hACE2 (pACE2 and mACE2, respectively), a cocktail of monoclonal antisera directed against DC-SIGN, DC-SIGNR, or both (DCSIGN), combinations of these antisera (pACE2 + DCSIGN) or an anti-MUC1 negative control antibody prior to inoculation with icSARS-CoV. Apical washes at the indicated time points were serially diluted and assayed by plaque assay on Vero E6 cells. Titers are represented by PFU/mL. Filled squares (black), polyclonal hACE2 only; filled triangles (blue), DC-SIGN/DC-SIGNR monoclonal cocktail; open square (pink), monoclonal and polyclonal hACE2; open circle (brown), no antibody; filled circle (red), monoclonal hACE2 only; open diamond (aqua), polyclonal hACE2 and DC-SIGN/DC-SIGNR monoclonal cocktail; open triangle (green), MUC 1.

Fig. 4

Growth kinetics of Urbani, icSARS-CoV and SARS-CoV GFP in Vero E6, MA104, and CaCo2 cells. To determine viral growth kinetics of the wild type and SARS-CoV GFP viruses, three cell types were infected: (A and D) Vero E6 (African green monkey, kidney); (B and D) MA104 (African green monkey, kidney); (C and D) CaCo2 (human, colorectal adenocarcinoma). Supernatant aliquots were harvested at the indicated times post-infection, serially diluted and titers assayed on Vero E6 cells. Titers are expressed as PFU/mL. Diamonds, Urbani; squares, icSARS-CoV; triangles, SARS-CoV GFP.

Fig. 5

SARS-CoV GFP infection of hamster airway epithelial cell cultures. (A) Ciliated airway epithelial cell cultures were derived from hamster and inoculated via the apical surface with SARS-CoV GFP (10⁶ PFU). GFP fluorescent images recorded 48 h post-infection are shown. Original magnifications: 10×. (B) Growth curves for icSARS-CoV and SARS-CoV GFP in hamster cultures were obtained from apical washes collected at 0.5, 1, 2, and 3 days post-infection. Samples were serially diluted and virus tittered by plaque assay on Vero E6 monolayers. All infections were performed in triplicate. (C) Square, icSARS-CoV; triangle, SARS-CoV GFP.