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 plaque reduction assay was performed as described by Leemans A. et al. [36] . Briefly, HEp-2 cells were seeded at a concentration of 17,500 cells/well in a clear 96-well plate (Falcon) 24 h prior to inoculation. Palivizumab was diluted 1/40 and further in a 1 2 dilution series, which was incubated with diluted virus for 1 h at 37 • C (5% CO 2 ). Afterwards, the cells were washed briefly with DMEM-0, and inoculated with 50 µL of the virus-antibody solution for 2 h at 37 • C (5% CO 2 ). Then, the inoculum was replaced with DMEM-10 containing 0.6% Avicel ®® (FMC biopolymer). The plates were incubated for three days at 37 • C (5% CO 2 ), washed with PBS and fixed with 4% paraformaldehyde solution. The cells were permeabilized with triton X-100, stained with palivizumab antibody followed by goat-anti-human IgG conjugated with HRP and coloured using chloronaphtol solution (Thermo Fisher Scientific). The neutralization titer was defined as the reciprocal of the highest antibody dilution producing 50% reduction in plaques (ED50), relative to virus-control wells without antibody.