Ab1128

Slides were fixed with PBS 1 × 0.5% Triton X-100 for 20 min and incubated with Fc Block (supernatant of 2.4G2 cells with 5% heat-inactivated human serum) for an additional 20  min. Cells were stained with 1:500 of goat anti-RSV antibody (Millipore Cat# AB1128, RRID: AB_90477) followed by 1:1000 of secondary rabbit anti-goat IgG (H + L)-HRP (Santa Cruz Biotechnology Cat# sc-2922, RRID: AB_656965) antibody. Antibody dilutions and washes were performed in 1X PBS. Nuclear staining was performed with Hoechst 33342 (ThermoFisher Scientific), and slides were mounted with glycerin for analysis under a fluorescence microscope (Olympus). Images were acquired using CellSens software (Olympus). Fluorescence intensity and quantification were measured using ImageJ.

Nasopharyngeal aspirate samples from 2 patients were diluted in 5 ml of DMEM low glucose containing 1000 µg/mL of gentamycin and inoculated into culture of Vero cells. After 1h incubation at 37 °C for complete viral adsorption, the viral inoculum was removed and added 10 ml of Opti-MEM supplemented with 2% of FBS and 1000 µg/mL of gentamycin into the flask. The culture was kept in an incubator at 37 °C and 5% CO₂ until macro-visualization of the viral cytopathic effect on the cells. Cells were harvested, centrifuged and the cell pellet was subjected to freeze-thaw cycles to obtain new viral particles. Afterwards, the viral aliquots were given sucrose cryoprotector and then stored in -80 °C until titration or further analysis. Viral titration was determined by plaque-forming unit (PFU) assay, using anti-RSV antibody (Millipore Cat# AB1128, RRID: AB_90477). The antigenic subgroup was identified by real-time PCR targeting the expression of the following primers and probes: Vi99990014_po (RSV A) and Vi99990015_po (RSV B) (ThermoFisher Scientific Cat# A39420). The viral nomenclature was made based on antigenic subgroup-city of isolation/strain number/year (RSV B-POA10/2018 and RSV A-POA43/2018).