Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection

Abstract

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

The efficiency of p27 peptide cleavage during in vitro respiratory syncytial virus (RSV) infection is cell line and RSV subtype dependent

“…We had previously observed differences in the kinetics of RSV infection by RSV strain and cell type (44), therefore, we speculated that the efficiency of p27 cleavage and the stability of the pre-F conformation may also be RSV strain and cell type dependent. For these studies we used Imaging Flow Cytometry to measure p27, Site Ø and Site II on virus-infected cells; we mitigated the contribution of fluorescence signal from any non-specific intracellular staining using masks to isolate signals to the surface of the cell membrane during data analysis.…”

“…These cell lines also differ in their post-translational modification and processing of the F protein, as higher measurable levels of p27 on HEp-2 cells indicated that this cell line was less efficient in cleaving p27 than A549 cells, confirming that HEp-2 and A549 cells elicit different responses to RSV infection. Our previous study demonstrated that HEp-2 cells produce higher cytokine anti-inflammatory responses to RSV infections, while A549 increases the gene expression of antiviral pathways (44). In addition to cell lines, we also demonstrated differences in p27 between RSV strains in RSV/A and RSV/B subgroups.…”