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

A stock solution of rHS was serially diluted 4-fold. Next, monolayers of non-immune (Vero, SH-SY5Y, SK-N-MC, 293T, HUEhT-1, Caco-2, HeLa, FLC-4, Huh7, FaDu, Detorit562, A549, HSQ89, NJG, JAR and JEG3) cells in 96-well plates were cultured with the diluted rHS samples at 35 °C. After a 4 day-incubation period, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Then, the RV-infected cells were detected by an indirect immunofluorescent assay using the mouse monoclonal antibody specific for the RV C protein and an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). The CCID 50 was calculated using the Spearman-Karber formulation 61, 62 . Immune (Raji, THP-1, U937, M8166, Jurkat, MT2) cells in 96-well plates (2.0 × 10 4 cells/well) were also cultured with the 4-fold diluted rHS samples for 4 days at 35 °C, and then fixed with 4% paraformaldehyde. The fixed cells were transferred to V-bottomed 96-well plates and centrifuged to remove the 4% paraformaldehyde fixing solution. The cells were then permeabilized with 0.5% Triton X-100. After removing the permeabilizing solution, indirect immunofluorescent staining was performed using the mouse monoclonal antibody specific for the RV C protein and an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). The cells were transferred to flat-bottomed 96-well plates to observe the RV-infected cells using a fluorescence microscope. To discriminate the fluorescent signals from the RV-infected cells from non-specific fluorescent signals, cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Lonza).