RSV can infect the human nasal epithelium via the basolateral route and shows distinct subgroup infectivity and basal cell tropism.

Respiratory syncytial virus (RSV) causes millions of lower respiratory tract infections (LRTIs) in young children, older adults, and immunocompromised populations every year. RSV infection initiates in the upper respiratory tract and can progress to the lower airways, resulting in bronchiolitis, pneumonia, and even death. RSV primarily infects epithelial cells apically, but we hypothesized that basolateral exposure of the respiratory epithelium could provide an alternative mechanism of infection that contributes to LRTI development. Using a human nose organoid-air-liquid interface (HNO-ALI) model, we performed apical and basolateral inoculations with contemporaneous RSV strains (RSV/A/Ontario [RSV/A/ON] and RSV/B/Buenos Aires [RSV/B/BA]) representing the two RSV subgroups (A and B) in both adult- and infant-derived HNO-ALIs. Basolateral RSV exposure resulted in delayed viral replication and apical release compared to apical infection. A statistically significant difference in basolateral infection frequency was observed between RSV/B/BA and RSV/A/ON (81.3% versus 25%). Basolateral infection selectively targeted a rare basal cell population, while preserving epithelial integrity. Using undifferentiated HNO-ALIs, we determined for the first time that Krt23+ activated basal cells are uniquely susceptible to RSV infection, a finding we confirmed in fully differentiated HNO-ALIs. Together, our findings show that RSV can infect the respiratory epithelium from the basolateral side by initially targeting a rare subset of basal cells before spreading apically to ciliated cells. Moreover, RSV/B/BA may have an advantage over RSV/A/ON in utilizing the basolateral infection route. These findings highlight an alternative RSV infection pathway and could be a potential mechanism for RSV spread to the lower airways.IMPORTANCEUnderstanding the pathogenesis of respiratory syncytial virus (RSV) is essential to understanding and preventing acute and long-term sequelae from infection. The canonical understanding of RSV infection is that the virus infects and is restricted to the apical ciliated cells upon inhalation or fomite exposure. We demonstrate that an alternative route of infection-the basolateral route-can be utilized by RSV to infect the apical ciliated cells of the respiratory epithelium. We also show for the first time a novel difference in infectivity between the two contemporaneous RSV strains (RSV/A/Ontario and RSV/B/Buenos Aires). In addition, we describe a rare basal subset-the Krt23+ activated basal cells-that are uniquely susceptible to RSV, expanding the known cellular tropism of RSV. Infection of basal cells can impact airway differentiation, homeostasis, and remodeling. Overall, our findings expand on RSV pathogenesis and indicate there are alternative mechanisms of infection and cell populations that are susceptible to RSV.