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.

This study was approved by the ethical committee of the Antwerp University Hospital and the University of Antwerp (16/46/491). Nasal secretions were collected from children showing symptoms of an RSV-related bronchiolitis during the winter seasons of 2016-2017 and 2017-2018 after written parental consent was given. The secretions were extracted by a nasal swab and/or a nasopharyngeal aspirate, which were stored at 4 • C for less than 10 h. One day prior to mucosal extraction, HEp-2 cells were seeded at a concentration of 17,500 cells/well in a clear 96 well plate (Falcon, Corning, NY, USA). Samples were vortexed for one minute with glass beads (Sigma-Aldrich; St. Louis, Mo, USA) before inoculating HEp-2 cells with 50 µL of a 1 4 dilution series of the sample, made in DMEM-0. After 2 h of incubation with the inoculum at 37 • C, 50 µL of DMEM containing iFBS, antibiotics (penicillin/streptomycin (Thermo Fischer Scientific), moxifloxacin (Sigma-Aldrich)) and anti-fungals (Fungizone)(Sigma-Aldrich) was added to obtain a final concentration of DMEM with 2% FBS. Plates were incubated for seven days at 37 • C (5% CO 2 ). After seven days, the plates were checked for syncytium formation and 50 µL of the well with the lowest concentration of original sample but still presenting CPE, was transferred to a newly seeded plate, following the previously described protocol. After an additional seven days, the wells were rechecked for syncytium formation. A total of 250 µL from wells presenting with syncytia was transferred to a freshly seeded T25, which was incubated until CPE was visible throughout the culture. Supernatant was collected, centrifuged for ten minutes at 1000× g, aliquoted, and snap frozen in liquid nitrogen (passage 0). Virus obtained from these clinical samples was propagated until passage 3 on HEp-2 cells to obtain a plaque forming unit (PFU) count high enough to perform the following experiments. One sample did not propagate efficiently on HEp-2 cells and was propagated for three passages on Vero cells until a high PFU was reached. Passage 3 cultures were tested for the following contaminants by qPCR: hMPV, Rhinovirus 1 and 3, PIV 1, 2, 3 and 4, adenovirus, Coronavirus 229E, NL63, OC43, Paraechovirus, Enterovirus 68 and Bocavirus and found negative for all. Several virus cultures tested negative on mycoplasma presence and no signs of bacterial or fungal presence was observed when the cultures were test grown in absence of antibiotics and anti-fungals.