SARS-CoV-2-infected human nasal epithelial air-liquid interface cultures

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, where the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly-infected and un-infected bystander cells and heightened in Alpha-infection. Alpha highly-infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2. Nasal epithelial ALI-cultures from three human adult and child donors (12-14 years) were infected with one of two strains of SARS-CoV-2 (ancestral strain VIC01, Alpha variant VIC17991). Single-cell RNA-sequencing with 10X Chromium coupled with Illumina sequencing was carried out with infected samples harvested at 72 hpi with ancestral strain (VIC01) and Alpha variant (VIC17991) and also 48 hpi of the ancestral strain (VIC01). Mock-controls were also sequenced. Count matrices were produced using Cellranger and the cells from the three donors (per age-group) were demultiplexed using genotyping information. Differential expression analysis using pseudo-bulking was carried out using edgeR and limma-voom.