A ciliated epithelial cell line as a model for respiratory syncytial virus research

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in infants and an increasingly recognized pathogen in the elderly. Conventional in vitro culture models for RSV rely on poorly differentiated cell lines that lack key airway epithelial features such as polarization, tight junctions and cilia, despite ciliated cells being the main RSV target. Primary airway epithelial cultures better reflect in vivo conditions, but they are difficult to access. We evaluated the human bronchial epithelial cell line BCi-NS1.1 differentiated at an air-liquid interface (ALI) as an alternative model. Three weeks after ALI, BCi-NS1.1 cultures exhibited advanced differentiation, including mature tight junctions, IL-13-induced MUC5AC production, a transepithelial potential difference of around −20 mV and 19% ciliated cells. These cultures expressed CX3CR1 receptor in cilia, which may serve as an entry point for RSV. These cultures were readily infected by RSV from the apical side, with infected cell numbers increasing from day 1 to day 5 post-infection. Apical viral titres reached ~2×10 5 PFU ml −1 between day 2 and day 4. Infection led to the formation of typical viral factories with inclusion-body-associated granules, apical release of new infectious virions and controlled shedding of infected cells. Because shedding limits subcellular analysis, we generated an RSV lacking NS2, a protein promoting cell rounding and shedding. Cells infected with RSV-Delta NS2 exhibited slower shedding and maintained their shape longer, enabling visualization of ribonucleoprotein migration towards and across the apical membrane. Overall, ALI-differentiated BCi-NS1.1 cells provide a suitable tool for studying epithelial responses to RSV infection.