A three-dimensional A549 cell culture model to study respiratorysyncytial virus infections
Background: Respiratory syncytial virus (RSV) is a primary cause of morbidity and mortality worldwide,affecting infants, young children, and immune-compromised patients; however, currently no vaccineis available for prevention of RSV infections. The overwhelming majority of our knowledge of how RSVcauses infection is based upon studies that have been carried out using traditional 2D methods, with cellscultured on flat plastic dishes. Although these simplified culture systems are essential to gain an insightinto the fundamentals of host-pathogen interactions, cells in 2D are not exposed to the same conditionsas cells in 3D tissues in the body and are therefore a poor representation of thein vivo microenvironment.In this study, we aim to develop the first 3D culture model for RSV infection using A549 cells to test itsutility for RSV pathogenesis.Methods: To generate spheroids, A549 cells were cultured using ultra-low attachment plates to generate25 × 103cell spheroids. The viability of the spheroids was assessed by trypan blue exclusion assay andflow cytometry showing prominent live cells throughout the spheroids confirming high viability overseven days of incubation.Results: Immunostaining of A549 spheroids inoculated with RSV, showed time-dependent disseminationof the viral antigen RSV-F within the spheroid, resulting in syncytia formation and a 3-fold increasein mucin secretion compared to the uninfected cells. Additionally, RSV successfully replicated in thespheroids producing infectious virus as early as day one post-inoculation and was sustained for up to 7days post-inoculation.Conclusions: Results show that A549 spheroids are susceptible and permissive for RSV since they exhibitthe characteristics of RSV infection including syncytia formation and mucin overexpression, suggestingthat A549 spheroids can be used a promising model for studying RSV in vitro.
Hassan zaraket, Nadia Soudani, Aya harb
Journal of Infection and Public Health ,DOI: https://doi.org/10.1016/j.jiph.2020.03.011, ISSN: 18760341, Volume: 13, Issue: 4, Pages Range: 1142-1147,