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.

Viral RNA was extracted from infected cell culture supernatants using the QIAmp viral RNA mini kit (Qiagen) according to the instructions provided by the manufacturer. Viral RNA of the G gene was transcribed to cDNA and amplified using the One-step RT-PCR kit (Qiagen) and the following primers as described by Houspie et al. [35] . For RSV-A, G267FW: 5 -ATG CAA CAA GCC AGA TCA AG-3 and F164RV: 5 -GTT ATC ACA CTG GTA TAC CAA CC -3 , for RSV-B, BGF: 5 -GCA GCC ATA ATA TTC ATC ATC TCT-3 and BGR: 5 -TGC CCC AGR TTT AAT TTC GTT C-3 were used. Primers were added to the reaction mix consisting of 10 µL 5× RT-PCR buffer, 2 µL dNTP, 2 µL enzyme, 20 µL H2O to a final amount of 30 pmol. Ten microliters (10 µL) of RNA extract was added to the reaction mix. The PCR was performed in a thermocycler (Unocycler, VWR; Radnor, PA, USA) with the following program: 30 min at 50 • C for the RT step, 15 min at 95 • C for PCR activation, 40 amplification cycles consisting of 30 s at 95 • C, 1 min at 55 • C and 1 min at 72 • C followed by a final extension step of 10 min at 72 • C. The length of the amplified cDNA was verified with 1% agarose gel electrophoresis and cDNA was visualized with Gelgreen™ (VWR). Amplified cDNA was delivered to the VIB Neuromics support facility (University of Antwerp) for PCR cleanup and DNA sequencing with the following primers as described by Houspie et al. [35] : in addition to the PCR amplification primers, for RSV-A: G516R Sequences were annotated in SnapGene and contigs were built in BioEdit with the CAP3 application. Multiple sequence alignments from reference strains and contigs, and phylogenetic trees were constructed in MEGA X using the maximum likelihood method.