Tu 80

RCASBP-(B)-CN-EGFP was kindly provided by Dr. Yao and Dr. Nair (Pirbright Institute). CRISPR/Cas9 vectors (5 µg) were mixed with Lipofectamine 2000 reagent (Thermo Fisher–Invitrogen) in Opti-Mem (Thermo Fisher–Invitrogen), and the mixture was applied to 1 × 10⁶ DF-1 cells. The mixture was replaced with DF-1 culture medium 6 h after transfection. One day after transfection we could detect green fluorescence in DF-1 cells, which indicated virus production. Cells were subpassaged, and the medium was changed 1 day after subpassaging. One day later, the medium containing virus was harvested and frozen at −70 °C until use. For viral infection, the medium containing virus was thawed at 37 °C and added to individual DF-1 and WL CEF clones. Four days post-infection, DF-1 and WL CEFs were observed using fluorescence microscopy (TU-80; Nikon, Tokyo, Japan) and analyzed using FACSCalibur (BD Biosciences, San Jose, CA, USA).

Cells were washed and concentrated on glass slides. After fixation in 2% paraformaldehyde for 15 min, the cells were incubated in a permeabilisation solution (0.1% Triton X-100 in PBS) for 10 min. Apoptotic cells were identified using an in situ Cell Death Detection Kit and TMR red (Roche Applied Science, Basel, Switzerland) that stains apoptotic cells red. Cells were counterstained with DAPI, mounted, and analysed under a fluorescence microscope (TU-80, Nikon).

The IFA assay was performed as previously described [68 (link)]. First, DF-1 cells were respectively infected with 200 TCID₅₀ rHPRS/JL3-1, rHPRS103, and rHPRS103-N123I at 37°C with 5% CO₂ for 2 h. After incubation, cells were washed three times with PBS at room temperature and kept in DMEM containing 5% (w/v) FBS at 37°C and 5% CO₂ for 72 h. Next, the cells were washed with cold PBST three times and fixed with cold solute ethanol for 15 min at room temperature. After washing three times, the cells were incubated with 4A3 (mouse anti-gp85 antibody) at a dilution of 1:200 in PBS for 1 h at 37°C. This was followed by washing and then incubation with 1:200 dilution of a fluorescein isothiocyanate-conjugated secondary antibody (FITC-conjugated goat anti-mouse IgG antibody) for 1 h at 37°C. After three washes with PBST, DF-1 cells were visualized using a fluorescence microscope (TU-80, Nikon, Tokyo, Japan). Normal DF-1 cells were used as negative controls.