Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication

Abstract

SUMMARY Background Differentiated human airway epithelial cell cultures have been utilized to investigate cystic fibrosis, wound healing, and characteristics of viral infections. These cultures, grown at an air-liquid interface in media with defined hormones and growth factors, recapitulate many aspects of the in vivo respiratory tract and allow for experimental studies at the cellular level. Objectives To optimize growth conditions for differentiated swine airway epithelial cultures, and to use these cultures to examine influenza virus infection and replication. Methods Primary swine respiratory epithelial cells were grown at an air-liquid interface with varying amounts of retinoic acid and epidermal growth factor. Cells grown with optimized concentrations of these factors for four weeks differentiated into multilayer epithelial cell cultures resembling the lining of the swine respiratory tract. Influenza virus infection and replication was examined in these cultures. Results/Conclusions Retinoic acid promoted ciliogenesis, whereas epidermal growth factor controlled the thickness of the pseudoepithelium. The optimal concentrations for differentiated swine cell cultures were 1.5ng/ml epidermal growth factor and 100nM retinoic acid. Influenza A viruses infected and productively replicated in these cultures in the absence of exogenous trypsin, suggesting that the cultures express a protease capable of activating influenza virus hemagglutinin. Differences in virus infection and replication characteristics found previously in pigs in vivo were recapitulated in the swine cultures. This system could be a useful tool for a range of applications, including investigating influenza virus species-specificity, defining cell tropism of influenza viruses in the swine respiratory epithelium, and studying other swine respiratory diseases.

The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells

“…We found that the apical surface of differentiated epithelial cells predominantly contains α2,6-linked sialic acid which is consistent with other reports1619. Although α2,3-linked sialic acids have been reported to be present on subepithelial cells of swALI cultures16, sialic acids of swine basal cells have not been analysed before. Here, we demonstrated that the KRT5 positive basal cells contain α2,3-linked sialic acids.…”

“…To this end, we and others studied IAV infection in differentiated ALI cultures of human airway epithelial cells which contain all major cell types present in human airway epithelium in vivo , namely, ciliated cells, secretory cells and basal cells12131415. Here we developed and employed swine ALI (swALI) cultures that can be used to analyze the infection by both human and swine IAV1617. Compared to the human counterpart, swALI cells have the advantage that the source of cells is well-defined and reproducible as far as the age and genetic background of the animals.…”

“…The growth conditions of swALI cultures were different from those described for human ALI cultures. The difficulty in establishing this system may explain why so far only few reports about differentiated respiratory epithelial cells of swine are available161718. The novel feature in our study is that we analysed events occurring late after infection not addressed in previous analyses with swine or human ALI cultures.…”

Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

“…ALI cultures from porcine airway cells have only recently been described for infection studies with viruses such as influenza virus and adenovirus2338 but not yet for bacterial infections. S. suis had been analyzed using a variety of immortalized respiratory epithelial cells1231323942, as well as polar porcine choroid plexus epithelial cells and human choroid plexus papilloma cells4546.…”

Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

“…Primary porcine airway epithelial cell cultures turned out to provide suitable model systems to investigate infection and replication of influenza viruses of avian, human, or swine origin (18)(19)(20)(21)(22)(23)(24)(25). Recent studies demonstrated that HA with a monobasic cleavage site is activated in these cultures without the need for exogenous trypsin (18,21,25), but the relevant proteases have so far not been identified or characterized in more detail.…”