Figure legends
Figure 1. Production and identification of pseudo-EBOV. (a) The complete genome of ZEBOV. (b) Detail of the ZEBOV G gene insertion in pcDNA4.0-G. The ZEBOV G gene was inserted through the BamH I site at the 5’ end and the Xba I site at the 3’ end, and the expression of the EBOV G gene was driven by the CMV immediate-early promoter (arrow). (c) Production strategy of pseudo-EBOV. After co-transfection with pcDNA4.0-G and pNL4-3.luc.RE, HEK293T cells were cultured at 37℃ with 5% CO2 for 24-36h, finally the pseudo-EBOV in supernatant was harvested. (d) Western blot analysis of pseudo-EBOV. HEK293T cells were transfected with two-plasmid system, or indicated plasmid, respectively. At 30h p.t., the transfected cells were harvested and lysed. Western blot was performed for indicated proteins by using corresponding antibodies. (e) Electron microscopy of pseudo-EBOV. The harvested pseudo-EBOV were applied to grids, stained with 1% sodium phosphotungstate, followed by observed and imaged using transmission electron microscopy. Pseudo-EBOV particles are indicated by red arrows. (f) Immunoelectron microscopy of pseudo-EBOV. The harvested pseudo-EBOV were applied to grids, followed by incubation with murine anti-EBOV GP monoclonal antibody for 1h at room temperature. After three washes, gold-labeled goat anti-mouse IgG was used as secondary antibody. Subsequently, the formvar-coated grids were stained with 1% sodium phosphotungstate after three washes. Images were acquired with transmission electron microscopy.
Figure 2. Infectivity and growth kinetics of pseudo-EBOV. (a) Cell tropism of pseudo-EBOV in a variety of cell lines was detected. As the positive control, pseudotyped vesicular stomatitis virus (VSV) was generated by the transfection of HEK293T cells with the VSV GP-encoding plasmid and pNL4-3.luc.RE. Data represent the mean relative luciferase units (RLUs) ± standard deviation (SD) from four parallel wells in 96-well culture plates. (b and c) Huh-7 cells were infected with pseudo-EBOV harvested at different post transfection time points. RLUs and titers of pseudo-EBOV were measured 48h later. Data represent the mean relative luciferase units (RLUs) ± standard deviation (SD) from four parallel wells in 96-well culture plates. (d) The pseudo-EBOV harvested at 36 h post transfection represents the pseudo-EBOV in first cycle, while the pseudo-EBOV harvested after 2 days after the first cycle of infection in Huh-7 cells represents the pseudo-EBOV in the second cycle. RLUs of pseudo-EBOV in the first and second cycle were measured. Data represent the mean relative luciferase units (RLUs) ± standard deviation (SD) from four parallel wells in 96-well culture plates. P-value was determined by unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, or no significance (n.s) (e) The titers of pseudo-EBOV in the first and second cycle were determined.
Figure 3. Neutralization assay for the evaluation of the neutralizing activity against EBOV. (a) Equine immunoglobulin fragments were subjected to pseudo-EBOV-based neutralization assay and live EBOV-based neutralization assay, respectively. The neutralizing titers of equine immunoglobulin fragment determined by the two assays are indicated. The average results of three independent experiments are presented. (b) Neutralizing activities of three different antibody-based samples were evaluated by pseudo-EBOV-based neutralization assay, negative horse IgG was used as negative control. The correlation between the neutralization efficiency and serial dilution of all samples is shown. The data represent three independent experiments.(c, d) NT50 and NT90 of the samples derived from pseudo-EBOV-based neutralization assay are shown. L.O.D., limit of detection. The mean ± standard deviations from three independent experiments are shown.