Single cell RNAseq of Influenza A replication kinetics in primary human bronchial epithelial cells

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. These data highlight the heterogeneous outcomes of influenza virus infections in primary human airway cultures and the disparate impacts of infected cell identity on burst size, even among preferentially infected cell types. NHBE cells were infected with Cal/04/09 at an MOI of 0.5 or mock infected (one well each). 10 hours post infection cells were washed and trypsinized to generate single cell suspensions. Single cell suspensions were filtered and counted and 10,000 cells per condition were used to generate Gel Bead-in Emulsions (GEMs). The 10X Chromium 3' GEX Capture kit and the 10X Chromium controller were used to generate GEMs and barcoding per manufacturers protocol (10X Genomics, Pleasanton, CA, USA). GEMs generated were used for cDNA synthesis and library preparation using the Chromium Single Cell 3' Library Kit and run on a NovaSeq S2. 2x100 PE Run.