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 RVFV pseudovirus packaging conditions were identified by using electron microscopy and western blotting. During the electron microscopy detection process, the harvested pseudoviruses or pseudovirus concentrates were first adsorbed to a copper grid and then stained with 1% phosphotungstic acid (Sigma, USA) for 2 to 3 min. After the residual staining solution was absorbed by using filter paper, the pseudovirus particles were observed by using an electron microscope. Additionally, the cell supernatant collected after transfection was mixed with 5× protein loading buffer (used for sodium dodecyl sulfatepolyacrylamide gel electrophoresis; Thermo) at a 4:1 dilution and added to a 1.5 mL centrifuge tube. After the samples were mixed thoroughly and boiled for 5 min, 15 μL of each sample was used for western blotting detection and analysis. During the experimental western blotting process, membranes were blocked with a 0.5% Tween-20 (Thermo) membrane-blocking solution containing 5% bovine serum albumin for 2 h at room temperature. The membranes were next incubated with a rabbit polyclonal anti-RVFV-MP12 primary antibody (1:4,000 dilution; IBT Bioservices, USA) for 2 h at room temperature and then with an HRP-labeled goat anti-mouse IgG secondary antibody (Invitrogen) for 1 h at room temperature. The blots were developed with a chemiluminescent developing solution (Thermo), and the experimental data were stored in the LAS-4000 system (Fujifilm, Japan).