Vero-H2 and helper 293–3-46-H2 cell lines were previously described [20 (link)]. Recombinant MVs were produced according to previously published procedures [20 (link)]. In brief, helper 293–3-46-H2 cells were transfected using calcium phosphate precipitation (ProFection kit, Promega, E1200) with two plasmids encoding for the MV genome and MV polymerase (pEMCLa). Three days after transfection, the helper cells were overlaid on Vero-H2 cells. Appearance of infectious centers was monitored by observing green fluorescent protein (GFP) expression under fluorescence microscope. Single viruses were then picked and propagated on Vero-H2 cells.
For virus stock preparation, Vero-H2 cells were infected at a multiplicity of infection (MOI) of 0.05 in OptiMEM (Life Technologies, 221705) for 2 h at 37 °C. DMEM-10 medium was then added on top and transferred to 32 °C until 95% of the cells expressed GFP. Cell culture media were removed and cells were scraped in OptiMEM. Viral particles were released by two freeze–thaw cycles. Titers of virus stocks were determined by 50% end-point dilution (tissue culture infectious dose 50, or TCID50) on Vero-H2 cells using the Spearman–Kärber method [30 ].
For virus stock preparation, Vero-H2 cells were infected at a multiplicity of infection (MOI) of 0.05 in OptiMEM (Life Technologies, 221705) for 2 h at 37 °C. DMEM-10 medium was then added on top and transferred to 32 °C until 95% of the cells expressed GFP. Cell culture media were removed and cells were scraped in OptiMEM. Viral particles were released by two freeze–thaw cycles. Titers of virus stocks were determined by 50% end-point dilution (tissue culture infectious dose 50, or TCID50) on Vero-H2 cells using the Spearman–Kärber method [30 ].
