Influenza A replication kinetics in primary human bronchial epithelial cells

Normal human bronchial epithelial cells (NHBEs) grown at air liquid interface form a pseudostratified epithelium in cell culture which recapitulates the environment in human upper airways. Here we investigated whether the cellular landscape of NHBEs impacts burst size and/or replication dynamics of influenza A virus. To determine whether replication kinetics of influenza A differ in ciliated versus secretory cells (two cell types for which Influenza A has tropism), two experiments were performed. In the first, NHBEs grown at air liquid interface were infected with Cal/07/09 (MOI=0.5) and were sorted into secretory and ciliated cells using FACS at 6 and 12 hours post-infection. Paired uninfected NHBEs were also sorted. 4 wells of NHBEs were infected or used as uninfected controls for each timepoint.The 2nd experiment was analogous to the first, except Cal/04/09 was used as the infection strain. 3 wells were infected (or left as uninfected control) this time. RNA sequenced with 150 bp paired-end reads on a NovaSeq. mRNA, cRNA, and vRNA levels for each influenza segment were quantified using InVERT (Phan et al., 2021; PMID 33658346). We found that ciliated cells produced higher counts of influenza A transcripts compared to secretory cells. However, proportions of mRNA, cRNA and vRNA to total segment RNA were not different between secretory and ciliated cells, indicating that flu replication kinetics proceed the same across these two important cell types. We also profiled interferon stimulated gene expression across different cellular subtypes in mock and influenza A-infected NHBE cultures using single cell RNAseq. Here, NHBE cells were infected with Cal/04/09 at an MOI of 0.5 or mock-infected. 10 hours post infection, cells were used to generated Gel Bead-in Emulsions for Chromium Single Cell 3' gene expression analysis. We found different profiles of ISGs across cell types, including many ISGs expressed in basal cells. 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.