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.

Syncytium formation has long been considered a typical characteristic of RSV infection in immortal cell lines, and it has been used as a measure of activity of the fusion protein [27] . HEp-2 cells were infected at a MOI of 0.05 and incubated for 48 h with an overlay of DMEM-10 containing 0.6% Avicel ®® to allow spreading of the infection to neighbouring cells only. Afterwards, cells were fixed, fluorescently stained and analysed with fluorescence microscopy. Mean syncytium size was determined by calculating the mean of the number of nuclei in the syncytia formed per sample, (Figure 6A ,B) as well as mean syncytium frequency ( Figure 6C ,D) by counting the number of nuclei belonging to syncytia relative to the total number of nuclei of infected cells. Mean syncytium size of all RSV-A clinical isolates ( Figure 6A ) lies between four and seven nuclei per cell, with BE/ANT-A1/16, BE/ANT-A8/17 and BE/ANT-A10/17 having the largest syncytia. The smallest syncytia were produced by BE/ANT-A12/17. Mean syncytium frequencies lie between 16% and 21%, with the lowest frequency found for BE/ANT-A10/17, which suggested that it promotes the formation of larger syncytia rather than many small syncytia ( Figure 6C ). Clinical isolate BE/ANT-B20/17 formed significantly larger syncytia with a mean size of 13 compared to all clinical isolates ( Figure 6B ). Reference strain RSV B1 formed almost no syncytia, with the smallest size and lowest frequency of all viruses tested. Viruses 2019, 11, x FOR PEER REVIEW 13 of 21 Figure 5 . Thermal stability profiles at 37 °C, 32 °C and 4 °C. Clinical isolates, RSV A2 and RSV B1 were aliquoted and exposed to 37 °C (A,B), 32 °C (C,D) or 4°C (E,F). One aliquot of each was snap frozen at 0 h, 24 h, 48 h and 72 h. Aliquots were used for quantification by conventional plaque assay and calculated to the amount at 0 h. Data represents mean values ± SEM (n = 3), significant differences compared to the reference strains are indicated by *p < 0.05;**p < 0.01; ***p < 0.001 (two-way ANOVA). produced by BE/ANT-A12/17. Mean syncytium frequencies lie between 16% and 21%, with the lowest frequency found for BE/ANT-A10/17, which suggested that it promotes the formation of larger syncytia rather than many small syncytia ( Figure 6C ). Clinical isolate BE/ANT-B20/17 formed significantly larger syncytia with a mean size of 13 compared to all clinical isolates ( Figure 6B ). Reference strain RSV B1 formed almost no syncytia, with the smallest size and lowest frequency of all viruses tested.