The simple act of breathing brings the human airway epithelium under assault by a variety of potentially pathogenic microbes. To maintain sterility, the airway epithelium exhibits sophisticated innate defense mechanisms that are both cytokine-mediated and physical or mechanical in nature. Despite these defenses, respiratory pathogens infect and produce disease in the human host. Their efficiency at doing so is underscored by the fact that acute respiratory illness is the most common infection reported in the human population (345). Parainfluenza viruses and influenza viruses are significant respiratory pathogens that target the ciliated airway epithelium for infection. The overall goal of this dissertation was to characterize infection of the human airway epithelium by wild-type parainfluenza and
influenza viruses and to define the ability of these viruses to negotiate host innate barriers against infection. Innate barriers presented by the human airway epithelium in vivo, are most closely recapitulated in vitro by cultures of well-differentiated, pseudostratified, ciliated airway epithelium. Utilizing this culture model system, in combination with both human and avian virus isolates, recombinant mutant viruses and live attenuated vaccine candidates, we conclude the following:
BOTH HPIV1 and HPIV2 EXHIBIT CILIATED CELL TROPISM: The human airway epithelium is composed of a heterogeneous cell population, including ciliated cells, mucus- producing cells and underlying basal cells. Viruses infecting the airway have been identified which individually target each of these populations. Adenovirus and Andes virus, for
example, infect basal cells and Clara or goblet cells, respectively (237, 259, 330), while many respiratory viruses target the ciliated cell for infection, including SARS-CoV, HPIV3 and RSV (284, 359, 360). HPIV1 and HPIV2 were demonstrated in this dissertation to also have ciliated cell tropism after apical inoculation with either virus, and also after basolateral inoculation for HPIV2 (data not shown). The ability to infect via the basolateral side
(equivalent to the serosal side of the epithelium in vivo) was previously described for the related measles virus (286) and may suggest that HPIV2 infection can be mediated through additional mechanisms, outside of direct binding to receptors at the apical surface.
Regardless of the route of inoculation, however, progeny HPIV1 and HPIV2 were shed apically, correlating with restriction of these viruses to the respiratory tract during infection in vivo.
Demonstration of ciliated cell tropism for HPIV1 and HPIV2 provides additional evidence that the ciliated cell plays an important role in the pathogenesis of
paramyxoviruses. The ciliated cell of the human airway epithelium is critical for lung function through its role in mediating mucociliary transport and is likely the predominant cell type throughout the conducting airways for this purpose. Whether this cell type is a common target for infection as a coincidence of receptor distribution or whether these viruses specifically target this cell type due to its abundance or ability to sense and respond to infection relative to other cell types present in the epithelium is unknown.
HPIV1 binds to α2,3-linked sialic acid, thus, tropism described here correlates with the cellular distribution of α2,3-linked sialic acid previously determined by lectin binding (303, 312). Notably, avian influenza viruses, which not do not infect the human upper respiratory tract with HPIV-like efficiently, also utilize α2,3-linked sialic acid and are
observed to infect ciliated cells in HAE (56, 256). However, the sialic acids bound by avian influenza viruses are distinct from the α2,3-linked sialic acids bound by HPIV with respect to
interaction with the adjacent glucosamine (9). Furthermore, avian influenza viruses that adapt for efficient infection of the human airway epithelium evolve to bind α2,6-linked sialic acid (9, 183). This adaptation correlates with evolution towards non-ciliated cell tropism for influenza viruses as described by Thompson et al. (2006) who determined cell types infected by a panel of human influenza viruses including pandemic-era isolates and recent strains (312). The mutations required to switch viral HA binding preference from α2,3- to α2,6-linked sialic acid (and thus, cell tropism from ciliated to predominantly non-cilated cells) have been described for certain HAs. With this information, we can now investigate how different cell types in HAE may respond to infection by the same pathogen. This can be achieved by mutation of the RBD in the same viral genetic background and assessment of parameters of infection such as cytokine production. Such studies may elucidate the
advantages of binding α2,6-linked sialic acid and/or infecting non-ciliated cells in the context of influenza virus infection and may also yield information that would be useful in
understanding why paramyxoviruses exhibit strict ciliated cell tropism.
BOTH HPIV1 AND HPIV2 BLOCK IFN PRODUCTION IN HAE: Detection of viral antigen in ciliated cells in the HAE model indicated that both HPIV1 and HPIV2 successfully infected the HAE culture system. In response to viral infection, the host often produces interferon, a cytokine that acts to establish an anti-viral state through the induction of transcription of numerous host genes (93). Since interferon can act to limit viral replication and subsequent spread, we also determined the growth kinetics and corresponding levels of IFN induced by these parainfluenza viruses in HAE. Previous work suggested that HPIV1 could induce a type I IFN response in MRC5 lung fibroblast cells, but not in A549 lung carcinoma cells, indicating that IFN production in response to HPIV1 infection was cell-type dependent (38, 324). Lack of detectable type I IFN in HAE in our experiments for either
wild-type HPIV1 or HPIV2 provided evidence that these viruses possessed robust interferon antagonists that were effective in ciliated cells.
IFN ANTAGONISM IS LINKED TO THE HPIV ACCESSORY PROTEINS WHICH