Packaging of Rift Valley fever virus pseudoviruses and establishment of a neutralization assay method

Li, Yuetao; Zhao, Yongkun; Wang, Cuiling; Zheng, Xuexing; Wang, Hualei; Gai, Weiwei; Jin, Hongli; Yan, Feihu; Qiu, Boning; Gao, Yuwei; Li, Nan; Yang, Songtao; Xia, Xianzhu

The plasmid used for transfection was extracted by using an endotoxin-free plasmid extraction reagent kit (Endo Free Plasmid DNA Mini Kit II; OMEGA, China). Twenty-four hours before transfection, 293T cells were inoculated onto 10-cm plates (Corning, USA) at a density of 4-5 × 10 6 cells/mL in 10 mL of Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Thermo, USA). The cells were then cultured at 37 o C with 5% CO 2 . When cell growth reached 80% to 90% confluence, 5 μg of the lentiviral plasmid pLV-EGFP-C (Invitrogen, USA), 3.75 μg of the PH1 plasmid (Invitrogen), and 1.25 μg of the recombinant plasmid pcDNA3.1-M-rvfv (the PH2 plasmid was used as a positive control) were transfected into the 293T cells by using Lipofectamine 2000 transfection reagent (Invitrogen). After the cells were cultured at 37 o C with 5% CO2 for 4 to 6 h, the culture medium was replaced with 10 mL of fresh 293T culture medium. After 24 h of transfection, the culture medium was replaced with 10 mL of fresh virus culture medium.