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...

Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø–specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F–specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø.

We constructed a human recombinant parainfluenza virus type 3 (rPIV3) that expresses enhanced green fluorescent protein (GFP) and used this virus, rgPIV3, to characterize PIV3 infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). The apical surface of HAE was highly susceptible to rgPIV3 infection, whereas only occasional cells were infected when virus was applied to the basolateral surface. Infection involved exclusively ciliated epithelial cells. There was little evidence of virus-mediated cytopathology and no spread of the virus beyond the ciliated cell types. Infection of ciliated cells by rgPIV3 was sensitive to a neuraminidase specific for ␣2-6-linked sialic acid residues, but not to a neuraminidase that cleaves ␣2-3-and ␣2-8-linked sialic acid residues. This provided evidence that rgPIV3 utilizes ␣2-6-linked sialic acid residues for initiating infection, a specificity also described for human influenza viruses. The PIV3 fusion (F) glycoprotein was trafficked exclusively to the apical surface of ciliated cells, which also was the site of release of progeny virus. F glycoprotein localized predominately to the membranes of the cilial shafts, suggesting that progeny viruses may bud from cilia per se. The polarized trafficking of F glycoprotein to the apical surface also likely restricts its interaction with neighboring cells and could account for the observed lack of cell-cell fusion. HAE derived from cystic fibrosis patients was not more susceptible to rgPIV3 infection but did exhibit limited spread of virus due to impaired movement of lumenal secretions due to compromised function of the cilia.