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.