Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology

Abstract

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.Human respiratory syncytial virus (RSV) is the most important viral agent causing serious pediatric respiratory disease worldwide (5). RSV infection causes common-cold-like symptoms that progress to lower respiratory tract disease in 25 to 40% of infected infants and results in hospitalization for 0.1 to 1.0% of those infected. Almost everyone has been infected by RSV by 2 years of age. The immunity induced by RSV infection typically is incomplete, and reinfection is common, although subsequent infections are partially restricted and the disease severity is reduced (5).There is a relative lack of direct, detailed information on the characteristics of RSV infection, spread, and pathology in the upper and lower respiratory tract of humans. RSV infects the superficial layer of the respiratory epithelium and has the capacity to spread throughout the conducting airways. However, the details of RSV infection remain unclear, including whether there is a cell specificity to infection, the extent of virus infection and its relationship to disease manifestation and severity, the extent and cause of tissue damage, and whether the formation of syncytia that are so prominent in nonpola...

The histopathology of fatal untreated human respiratory syncytial virus infection

“…This suggests a tropism for cells of the distal airway, and more homogeneous cell populations in lower airways. As expected, 13 ciliated cells are infected, and it appears that nonciliated cells are also infected as noted in an adenoid organ culture model. 14 Basal cells appear to distinctly be not infected (Figure 1a).…”

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus–host interactions

“…Our results suggest that aMPV infection takes place at the apical region of the epithelium, which is comparable to the observations made after RSV infection of human nasal, tracheobronchial and bronchiolar airway epithelial cells (Zhang et al, 2002). Cilia may allow the virus to attach and therefore it may be suggested that polarized epithelial cells are the main target cells of aMPV (Jones et al, 1986;O'Loan & Allan, 1990;Majó et al, 1995;Jirjis et al, 2001;Zhang et al, 2002). In the present state of knowledge, the oviduct epithelium is the only other surface susceptible to aMPV, apart from respiratory epithelia (Jones et al, 1988).…”

“…A similar observation was seen in one study case of RSV infection of respiratory epithelial cells showing loss of ciliary activity without apoptotic signs (Domachowske et al, 2001). The tissue damage after RSV infection is suggested to be due to other destructive immune responses, induced to control virus replication (Zhang et al, 2002).…”

Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells

“…RSV replicates in these cells with relatively little evidence of cytopathology, contrary to the extensive morphologic changes observed in infected nonpolarized epithelial cell lines, such as HEp-2 cells. Furthermore, RSV infection is limited to the most superficial layer of ex vivo cultured stratified human respiratory epithelium (11). These findings are consistent with many clinical aspects of RSV infection.…”

“…In vitro and ex vivo studies have shown that the virus exhibits a preference for apical budding from infected, polarized epithelial cells (10,11). RSV replicates in these cells with relatively little evidence of cytopathology, contrary to the extensive morphologic changes observed in infected nonpolarized epithelial cell lines, such as HEp-2 cells.…”