Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures

Human respiratory syncytial virus (HRSV) is the leading cause of bronchioli- tis in infants. Two subgroups of HRSV (A and B) routinely cocirculate. Most research has been performed with HRSV-A strains because these are easier to culture than HRSV-B strains. In this study, we aimed to compare the replicative fitness and HRSV-induced innate cytokine responses of HRSV-A and HRSV-B strains in disease-relevant cell culture models. We used two recombinant (r) clinical isolate-based HRSV strains (A11 and B05) and one recombinant laboratory-adapted HRSV strain (A2) to infect commercially avail- able nasal, bronchial, and small-airway cultures. Epithelial cells from all anatomical loca- tions were susceptible to HRSV infection despite the induction of a dominant type III interferon response. Subgroup A viruses disseminated and replicated faster than the subgroup B virus. Additionally, we studied HRSV infection and innate responses in air- way organoids (AOs) cultured at air-liquid interface (ALI). Results were similar to the commercially obtained bronchial cells. In summary, we show that HRSV replicates well in cells from both the upper and the lower airways, with a slight replicative advantage for subgroup A viruses. Lastly, we showed that AOs cultured at ALI are a valuable model for studying HRSV ex vivo and that they can be used in the future to study factors that influence HRSV disease severity. IMPORTANCE Human respiratory syncytial virus (HRSV) is the major cause of bron- chiolitis and pneumonia in young infants and causes almost 200,000 deaths per year. Currently, there is no vaccine or treatment available, only a prophylactic mono- clonal antibody (palivizumab). An important question in HRSV pathogenesis research is why only a fraction (1 to 3%) of infants develop severe disease. Model systems comprising disease-relevant HRSV isolates and accurate and reproducible cell culture models are indispensable to study infection, replication, and innate immune responses. Here, we used differentiated AOs cultured at ALI to model the human air- ways. Subgroup A viruses replicated better than subgroup B viruses, which we spec- ulate fits with epidemiological findings that subgroup A viruses cause more severe disease in infants. By using AOs cultured at ALI, we present a highly relevant, robust, and reproducible model that allows for future studies into what drives severe HRSV disease.