We have investigated RSV infection in a model of primary WD HAE cells by using a recombinant RSV that expresses GFP to monitor RSV infection in living cells. We found that rgRSV primarily, perhaps exclusively, targets the lumenal ciliated columnar airway epithelial cells via the apical surfaces of the cultures. In contrast, rgRSV infection was not observed after basolateral inoculation and was not enhanced by disruption of epithelial tight junctions or by mechanical damage to the tissue. These observations suggest that basal cells and the basolateral surfaces of columnar cells are not permissive for rgRSV infection. Progeny rgRSV were released from the apical surfaces of WD HAE cultures, and rgRSV infection spread via the lumenal surface to neighboring ciliated cells. A previous study showed that RSV was released from the apical membranes of polarized Vero cell monolayer cultures (34
). However, since these cells are not derived from human tissue, are not ciliated, are immortalized, and form a single monolayer, it was important to confirm and extend these results in a primary, differentiated, multilayered, ciliated HAE culture. An interesting finding, only possible in a culture model such as that used in the present study, was that virus spread in HAE cells appeared to be affected by the directionality of the cilial beat. The ciliary beat has been shown to direct the movement of the periciliary fluid as well as the overlying mucus in this culture model (26
A striking observation was that rgRSV appeared to primarily, and perhaps exclusively, infect ciliated cells. This was not only true in WD cultures but was also observed in immature HAE cultures undergoing differentiation, where susceptibility to rgRSV infection was dependent on differentiation and coincident with ciliogenesis. This may mean that the cilia themselves are directly necessary for rgRSV infection and might, for example, be the site of viral attachment. A direct role of cilia in RSV infection would be an important difference from infection of established cell lines, such as HEp-2 cells, which are highly permissive to RSV but are not ciliated. Even if cilia are not directly involved with RSV infection, the maturational requirement of primary HAE cells for susceptibility to rgRSV infection is interesting and contrasts with the ability of RSV to infect many established cell lines. One possibility is that infection of established cell lines proceeds by an alternative pathway, one that might not be representative of that which occurs for the airway epithelium in vivo. For example, it will be of interest to test whether the requirement for glycosaminoglycans for efficient RSV infection of established lines, such as HEp-2 cells, is also a requirement in the WD HAE cultures. WD HAE cultures likely will be the cell model of choice for examining the mechanisms of virus-host cell interactions important for initiating RSV infection, at least for the airway epithelium.
RSV infection initiates in the upper airway regions, after which the virus can spread to more distal airway regions and to the alveoli (11
). Our data indicate that the characteristics of infection are similar for epithelial cell cultures derived from upper and lower airway epithelia, specifically the nasal, tracheobronchial, and bronchiolar regions. We did not test the ability of rgRSV to infect cells of the alveolar region, and thus, the observations that we describe apply strictly to the epithelium of the conducting airways. We found that for culture models from these airway regions, the extent of infection correlated with the number of ciliated cells present in the culture. Although ciliated cells of the upper airways differ in height and length of the cilial shaft from ciliated cells of the bronchioles, we found that both upper and lower ciliated cell types were susceptible to rgRSV infection.
Two clinical strategies currently in use against RSV infection, namely, an anti-F protein monoclonal antibody used in pediatric immunoprophylaxis and the nucleoside analog ribavirin used in RSV therapy (33
), were efficacious against rgRSV infection in WD HAE cultures. Our experiments emphasized important aspects of the polarity of airway epithelium. The antibody was only effective at preventing rgRSV spread when presented on the apical surface. This finding suggests that trans-epithelial cell transport of this IgG antibody from the basolateral to the apical compartment was not efficient in vitro, at least not within the incubation period studied. It is also consistent with clinical experience: serum IgG antibodies are inefficiently transported to the lumenal surface of the respiratory tract, and RSV-neutralizing serum antibodies restrict RSV infection only when present at high titer (10
). In contrast, ribavirin was active with basolateral application mimicking parental administration in vivo.
The ability of ribavirin to strongly inhibit RSV replication in vitro had been shown previously with nonpolarized cells (16
). In the present work, the strong inhibition of GFP expression observed with ribavirin, added immediately before virus inoculation, shows that this nucleoside analog readily achieved bioavailability and greatly restricted gene expression. In addition, ribavirin acts early in the viral infection cycle, greatly restricting viral gene expression in addition to blocking the spread of infection.
An interesting finding of the present study was the observation that GFP expression was maintained in WD HAE cultures for up to 3 months, the longest period during which the cultures were maintained. It is not known whether the GFP-expressing cells observed after 3 months were ones that had been infected at the outset and maintained throughout the entire period or whether there was turnover of cells and reinfection of newly differentiated ciliated cells. Since the half-life of ciliated airway cells in these cultures has been estimated to be on the order of 40 to 100 days (1
), it seems likely that some, and perhaps most, of the cells observed at 3 months were not among the infected cells present during inoculation. It is unknown whether rgRSV infection alters the half-life of ciliated airway cells in this culture system. However, it is clear that rgRSV infection produced little or no virus-specific obvious cytopathic effect in WD HAE cells. In particular, there was no evidence of syncytium formation in either columnar, intermediate, or basal cells. Thus, while rgRSV infection might have subtle effects that were undetected, such as altering cellular half-life or inducing apoptosis, on a gross level rgRSV infection in this model of WD HAE was noncytopathic. We confirmed that this was not an artifact of rgRSV, since similar results were obtained with its wild-type recombinant parent lacking GFP as well as with a biologically derived wild-type RSV. The last two viruses have been shown to cause respiratory disease in chimpanzees, confirming that they are wild-type viruses (40
In contrast to rgRSV, influenza A virus infection of WD HAE cultures resulted in rapid and extensive obvious cytopathology. This is consistent with clinical pathological findings that influenza virus infection results in extensive damage to the respiratory epithelium in infected humans (14
). These findings suggest that the tissue damage that is observed in the ciliated airway epithelium in vivo in response to RSV infection might not be due directly to viral presence and growth but rather is a consequence of destructive components of a robust immune response that eventually clears the virus infection. The situation was very different with influenza A virus, where virus infection was inherently highly cytopathic even in the absence of an immune response. Since we did not examine rgRSV infection in a system that corresponds to the alveolar epithelial region, it is possible that viral cytopathology might be different in that compartment.
The observed absence of obvious cytopathology by RSV infection in our culture model is in contrast to a previous report that also used a polarized, ciliated HAE cell culture model to assess the effect of wild-type RSV infection (37
). In that study, RSV infection was found to have multiple effects: (i) lumenal cilial beat frequency was significantly inhibited within 2 h of virus addition to the lumenal surface, (ii) loss of cilia occurred 24 h after infection, (iii) syncytia formed, and (iv) all ciliated cells were sloughed from the culture by 3 to 5 days postinfection. Under similar conditions with our culture system, we did not observe any of these effects with rgRSV or with a wild-type RSV. The differences between these studies remain to be explained and may involve differences in the virus or cell culture systems used.
The lack of obvious RSV cytopathology with the HAE culture system described in this study is probably related to the lack of syncytium formation. Most studies of RSV have been performed with nonpolarized epithelial cell lines (e.g., HEp-2) that undergo cell-cell fusion 2 to 5 days after exposure to RSV and ultimately undergo cell death. However, using our polarized WD HAE model, no syncytia were ever observed. In vivo, syncytia have not been a prominent pathological feature of RSV infection, although they have been observed in autopsy tissue from RSV-infected patients (28
), and RSV can cause giant-cell pneumonia in immunocompromised individuals (8
). It might well be that, like our WD HAE cultures, the epithelium of the airways is refractory to syncytium formation and that which is observed in vivo involves the alveolar epithelium. In a study with the polarized epithelial cell line Vero C1008, Roberts et al. (34
) reported that RSV matured at the apical surface without syncytium formation. They also found the F protein preferentially expressed at the apical surfaces of RSV-infected polarized cell monolayers, and it seems likely that the same is true of the multilayer HAE cultures in the present work. If the F protein is distributed exclusively at the apical surface, its interaction with adjacent cells likely would be restricted, and therefore the formation of syncytia would be suppressed. This segregation might not be an all-or-none phenomenon, as suggested by the data of Roberts et al. The efficiency of polar segregation might be further compromised under certain conditions and could be responsible for the syncytium formation observed by Tristram et al. (37
). It also might be that the efficiency of polar segregation could be rendered less efficient in vivo, perhaps during an immune attack, which could account for the syncytia that are sometimes observed in vivo.
The ability of RSV to transfer genes to the ciliated cells of the airway epithelium after lumenal delivery indicates that this virus may provide a new vector system suitable for disorders of the lung epithelium, such as CF lung disease. In the case of CF, the ciliated airway epithelial cells are considered to be the target cell type that requires correction (2
). So far, gene therapy strategies have investigated vector systems such as adenovirus types 2 and 5, adeno-associated virus type 2, and retroviruses, none of which efficiently transfer genes to the lumenal columnar airway epithelial cells. The inefficiency of gene transfer is reported to be due to a number of barriers that are present at the apical surface of the lung epithelium, most notably the absence of receptors for the specific viruses from the apical surfaces of the cells (31
) and the presence of a restrictive glycocalyx barrier (30
). For RSV, neither of these potential barriers appears to be restrictive for efficient infection.
Other vector systems have been reported to deliver genes to columnar airway cells via the lumenal membrane. Sendai virus vectors have been shown to infect ciliated cells of the rodent airway (43
), but the efficiency with which this rodent virus infects HAE cells remains to be determined. Lentiviral vectors pseudotyped with components of Ebola virus membrane proteins have been shown to efficiently transduce HAE cells in vitro, suggesting that combining the efficiency of this system with the potential long duration of lentivirus-mediated gene expression may provide an effective vector for gene therapy strategies for the lung epithelium (19
). Interestingly, lentivirus pseudotyped with RSV F and G proteins did not transduce HAE cells, although there is some question as to the efficiency of virion formation with this pseudotyping strategy (19
). The present study confirms that RSV indeed targets the lumenal epithelial cells. Coronavirus (39
) and adeno-associated virus type 5 (45
) have also been reported to be efficient for gene transfer to the lung epithelium. Further studies are required to determine the efficacies of these vectors for potential clinical applications.
The high efficiency of RSV infection in WD HAE cultures; the specificity of infection for the lumenal ciliated cells; the unexpected long duration of transgene expression, at least in the absence of an immune response; and the lack of obvious cytopathology in infected cultures indicate that RSV is an attractive candidate for further development as a gene transfer vector to target ciliated airway epithelium. Of course, further modification of the recombinant virus likely will be necessary for this application. For example, it will be necessary to use a version of recombinant RSV that does not cause respiratory tract disease. Fortunately, a wide array of recombinantly derived attenuated versions of RSV are available, and there is extensive clinical experience with experimental RSV vaccines that will aid in the development of vectors suitable for gene transfer to the lung epithelium.