Oxoid purified agar

For generating recombinant viruses co-cultivated HEK293-T cells and MDCK-II cells were co-transfected with the eight pHW-2000 plasmids derived from WSN (WT or mutant) using Lipofectamin™ 2000 (Invitrogen). After 6 h of incubation in Gibco™ Opti-MEM™ I Reduced Serum Medium (Thermo Fisher) at 37 °C the supernatant was removed and cells were incubated with infection medium (DMEM supplemented with 0.7% BSA, 1% CaCl₂/MgCl₂, and TPCK-Trypsin) for 48 h at 37 °C. The virus rescue supernatant was plaque purified on MDCK-II cells by plaque assays using Oxoid™ purified agar (Thermo Fisher). Then, MDCK-II cells were infected with single plaques diluted in infection PBS and incubated in infection medium for 48 h at 37 °C. Final virus stocks were obtained by passaging this supernatant once on MDCK-II cells with a defined MOI of 0.001. To control for the respective mutation, MDCK-II cells were infected with an MOI of 2. After 6 h, cells were lysed and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). The respective gene segment was amplified using OneStep RT-PCR Kit (Qiagen) and sequenced by Sanger sequencing (Eurofins Genomics).

To titrate the recombinant vaccinia viruses, confluent monolayers of BSC40 cells were infected with different dilutions of the viral stock starting from 1:10 to 1:1,000,000 in phosphate-buffered saline (PBS) for an hour to allow the virus to attach to the cells. After an hour, cells were overlaid with MEM that contained 2% Oxoid agar (Oxoid purified agar; Thermo Scientific). Cells were incubated at 37°C for 2 days and then fixed. Cells were stained with crystal violet solution (Sigma-Aldrich), and plaques were counted. Titration of VSV-LASV was done in a similar manner using Vero.E6 cells.