A three-dimensional A549 cell culture model to study respiratory syncytial virus infections

Fatima Saleh; Aya Harb; Nadia Soudani; Hassan Zaraket

doi:10.1016/j.jiph.2020.03.011

A three-dimensional A549 cell culture model to study respiratory syncytial virus infections

J Infect Public Health. 2020 Aug;13(8):1142-1147. doi: 10.1016/j.jiph.2020.03.011. Epub 2020 Apr 29.

Authors

Fatima Saleh¹, Aya Harb², Nadia Soudani³, Hassan Zaraket⁴

Affiliations

¹ Department of Medical Laboratory Sciences, Faculty of Health Sciences, Beirut Arab University, Lebanon.
² Faculty of Medicine, Department of Experimental Pathology, Immunology & Microbiology, American University of Beirut, Beirut, Lebanon.
³ Faculty of Medicine, Department of Experimental Pathology, Immunology & Microbiology, American University of Beirut, Beirut, Lebanon; Faculty of Medicine, Center for Infectious Diseases Research, American University of Beirut, Beirut, Lebanon; Doctoral School of Sciences and Technology, Research Platform for Environmental Science (PRASE), Faculty of Sciences, Lebanese University, Lebanon.
⁴ Faculty of Medicine, Department of Experimental Pathology, Immunology & Microbiology, American University of Beirut, Beirut, Lebanon; Faculty of Medicine, Center for Infectious Diseases Research, American University of Beirut, Beirut, Lebanon. Electronic address: hz34@aub.edu.lb.

Abstract

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 vaccine is available for prevention of RSV infections. The overwhelming majority of our knowledge of how RSV causes infection is based upon studies that have been carried out using traditional 2D methods, with cells cultured on flat plastic dishes. Although these simplified culture systems are essential to gain an insight into the fundamentals of host-pathogen interactions, cells in 2D are not exposed to the same conditions as 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 its utility for RSV pathogenesis.

Methods: To generate spheroids, A549 cells were cultured using ultra-low attachment plates to generate 25 × 10³ cell spheroids. The viability of the spheroids was assessed by trypan blue exclusion assay and flow cytometry showing prominent live cells throughout the spheroids confirming high viability over seven days of incubation.

Results: Immunostaining of A549 spheroids inoculated with RSV, showed time-dependent dissemination of the viral antigen RSV-F within the spheroid, resulting in syncytia formation and a 3-fold increase in mucin secretion compared to the uninfected cells. Additionally, RSV successfully replicated in the spheroids producing infectious virus as early as day one post-inoculation and was sustained for up to 7 days post-inoculation.

Conclusions: Results show that A549 spheroids are susceptible and permissive for RSV since they exhibit the characteristics of RSV infection including syncytia formation and mucin overexpression, suggesting that A549 spheroids can be used a promising model for studying RSV in vitro.

Keywords: 3D culture; A549; Infection; Mucin; Respiratory syncytial virus (RSV).

MeSH terms

A549 Cells
Animals
Cell Culture Techniques
Chlorocebus aethiops
Humans
In Vitro Techniques
Respiratory Syncytial Virus Infections* / pathology
Respiratory Syncytial Virus, Human
Vero Cells