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

To confirm the multiplicity of infection of the pseudoviruses and to obtain a stable infection effect, we measured the RVFV pseudovirus titers. Specifically, the harvested and purified virus solution was used to determine the viral titers (although frozen virus solution could have been used). Notably, very large differences were detected in the viral titers measured using different cell lines and methods; thus, very large differences might have existed between the viral titer values and the viral titers required to infect target cells. To avoid this issue, the same virus solution-based measurement method was used to determine the viral titers in order to provide more accurate measurement of the virus packaging effects. Next, 293T cells were cultured to the logarithmic growth phase and dissociated with 0.25% trypsin (Gibco). After counting, the cells were seeded into 96-well culture plates at a density of 5 × 10 4 cells/well and cultured at 37 o C with 5% CO 2 . For virus inoculation, pseudovirus solution stored in a −80 o C freezer was thawed in an ice bath and then subjected to a series of gradient dilutions using DMEM containing 10% FBS (10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 , 10 −7 , 10 −8 , and 10 −9 ). Each dilution was added to three replicate wells. After the cells were cultured at 37 o C with 5% CO 2 overnight, the culture medium was replaced with 100 μL of DMEM containing 10% FBS, and the cells were continuously cultured for 48 to 72 h. The cells were then observed via fluorescence microscopy, and the number of cells with positive green fluorescence in each well was calculated. The virus titer was the number of cells with positive green fluorescence multiplied by the corresponding fold dilution.