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.

HEp-2, A549 and BEAS-2B cells were seeded at a concentration of 17,500 cells/well in black CELLSTAR ®® 96 well plates with a µclear ®® flat bottom suitable for fluorescence microscopy (Greiner-bio one) one day prior to inoculation. Briefly before inoculation, the cells were washed with DMEM-0. Clinical RSV and RSV A2 were diluted to infect the cells at a multiplicity of infection (MOI) of 0.01. Virus was left to adhere for 2 h at 37 • C, 5% CO 2 and replaced with DMEM-10. Cells were fixed with 4% paraformaldehyde after 24 h, 48 h and 72 h, permeabilized with triton X-100 and stained with polyclonal goat-anti-RSV antibody (Virostat; specificity all viral antigens) followed by donkey-anti-goat secondary antibody conjugated with Alexa Fluor 488 (AF488) (Thermo Fisher Scientific) and additional DAPI nucleus staining (Sigma-Aldrich).