Isolation and Characterization of Clinical RSV Isolates in Belgium during the Winters of 2016–2018

Van der Gucht, Winke; Stobbelaar, Kim; Govaerts, Matthias; Mangodt, Thomas; Barbezange, Cyril; Leemans, Annelies; De Winter, Benedicte; Van Gucht, Steven; Caljon, Guy; Maes, Louis; De Dooy, Jozef; Jorens, Philippe; Smet, Annemieke; Cos, Paul; Verhulst, Stijn; Delputte, Peter L.

The HEp-2, A549 and Vero cell lines were obtained from and cultured to the instructions of ATCC. The BEAS-2B cell line was a generous gift from Dr. Ultan F. Power (Queens University Belfast, Ireland). All cells were cultured in Dulbecco's modified Eagle medium containing 10% inactivated fetal bovine serum (DMEM-10) (Thermo Fisher Scientific, Waltham, MA USA). RSV reference strains A2 and B1 were obtained from BEI resources, RSV A2 was cultivated in HEp-2 cells as described by Van der Gucht W. et al. [32] and RSV B1 was cultivated on Vero cells in medium containing 2% inactivated foetal bovine serum (iFBS) until cytopathic effects (CPE) were visible throughout the flask. Virus was collected as described for RSV A2 and quantified in a conventional plaque assay on HEp-2 cells as described by Schepens et al. [33] . Briefly, HEp-2 cells were seeded at a concentration of 17,500 cells/well in clear 96 well plates (Falcon) one day prior to infection. Cells were washed with DMEM without iFBS (DMEM-0) and infected with 50 µL of a 1/10 dilution series made in DMEM-0. Cells were incubated for 2h at 37 • C after which the inoculum was replaced by DMEM-10 containing 0.6% Avicel ®® (FMC biopolymer) and incubated for three additional days at 37 • C (5% CO 2 ). Afterwards, cells were washed with PBS, fixed with 4% paraformaldehyde solution and stained with palivizumab (leftovers provided by the Department of Paediatrics, Antwerp University Hospital) and goat-anti human secondary IgG conjugated with horseradish peroxidase (HRP) (Thermo Fisher Scientific) and visualized using chloronaphtol solution (Thermo Fisher Scientific).