Background
Respiratory co-infections caused by HRSV and SARS-CoV-2 often result in severe respiratory disease, but the underlying cellular/molecular mechanisms are poorly understood. Using an in vitro model of HRSV/SARS-CoV-2 co-infection, we investigated how individual cells respond to simultaneous or single infections, providing insights into synergistic/antagonistic viral interactions.
Methods
Single-cell RNA sequencing (scRNA-seq) data of the A549-ACE2-expressing cell line, co-infected with HRSV and SARS-CoV-2 (MOI: 0.01) were analyzed 72 hours post infection. The expression of SARS-CoV-2 and HSRV-specific genes was examined simultaneously with cellular genes to determine the presence or co-presence of the viruses in each profiled cell. Gene expression profiles of single/co-infected cells were used to identify specific cell markers and make functional inferences about the different cell states.
Results
ScRNA-seq data identified four distinct cell clusters: 1) uninfected, 2) SARS-CoV-2-infected, 3) HRSV-infected, and 4) SARS-CoV-2/HRSV co-infected cells. Within Cluster 4, two sub-clusters were further distinguished based on HRSV gene expression levels: 4a (low) and 4b (high). Marker analysis revealed molecular signatures of both protein-coding and long non-coding (lnc) RNA characterizing each of the four cell clusters. Specifically, co-infected cells showed altered expression of genes linked to viral defense (n=27; P=6.2×10⁻⁸), innate immunity (n=30; P=2.5×10⁻⁵) and type I IFN regulation (n=8; P=3.4×10⁻⁴). When analyzing sub-clusters, JAK-STAT (n=8; P=5.7×10-5) and ERK1/ERK2/MAPK (n=15; P=9.3×10-5) signaling pathways were specifically up-regulated in sub-cluster 4a and 4b, respectively. Co-infected cells also showed higher expression of at least 16 lncRNAs, some not yet functionally annotated, while others (e.g. AC011287.1) may be involved in trained immunity. As for the sub-clusters, lncRNAs NEAT1 and MALAT1 were downregulated in 4a compared to 4b (P=1.1×10-9 and P=2.3 x 10-16, respectively).
Conclusions
ScRNA-seq revealed transcriptionally distinct subpopulations of lung epithelial co-infected cells. Two sub-clusters of coinfected cells, characterized by different HRSV expression levels, are presumably associated with a competitive/cooperative interaction between HRSV and SARS-CoV-2 within the same cell. Further analyses will be mandatory to ascertain the functional role exerted by lncRNAs that may have therapeutic relevance in the context of HRSV/SARS-CoV-2 coinfection.