Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

Sakata, Masafumi; Tani, Hideki; Anraku, Masaki; Kataoka, Michiyo; Nagata, Noriyo; Seki, Fumio; Tahara, Maino; Otsuki, Noriyuki; Okamoto, Kiyoko; Takeda, Makoto; Mori, Yoshio

Neutralization assays for VSV FLuc -RV/CE2E1 and VSV FLuc -G. Goat antiserum against RV and an unimmunized normal goat serum were 4-fold serially diluted in DMEM. VSV FLuc -RV/CE2E1 and VSV FLuc -G, which express FLuc, were mixed with the diluted sera and incubated for 1 h at 4 °C. Then, the Vero cell monolayers in 96-well plates were infected with VSV FLuc -RV/CE2E1 and VSV FLuc -G pretreated with the sera. At 24 h p.i., the RLU from the cells was measured using the Bright-Glo Assay System and POWER SCAN HT. Analysis of calcium dependency in pseudotype virus infections. Calcium dependency in the pseudotype virus infections was assessed using a method similar to that reported previously 35 . Vero cells in 96-well plates were incubated with VSV GFP -RV/CE2E1 and VSV GFP -MV/FH, which express GFP, for 2 h at 4 °C, and then washed with pre-chilled DPBS, twice. The cells were then cultured in DMEM free from CaCl 2 (Thermo Fisher Scientific) or containing various concentrations of CaCl 2 for 1 h at 37 °C. The cells were then cultured in DMEM containing 2 mM CaCl 2 and 10% FBS at 37 °C. At 24 h p.i. the number of GFP-expressing cells, which correspond to VSV GFP -RV/CE2E1-or VSV GFP -MV/FH-infected cells, was counted using a fluorescence microscope. Genome copy number measurements for VSV GFP -RV/CE2E1, VSV GFP -G, and VSV GFP -∆G. The genome copy number for VSV GFP -RV/CE2E1, VSV GFP -G, and VSV GFP -∆G was analyzed by reverse transcription-quantitative PCR (RT-qPCR) using a set of primers specific for the VSV N gene, as reported previously 19 .