3.3 Infectivity screening and kinetics of pseudotyped EBOV
To figure out a susceptible cell line of pseudotyped EBOV for subsequent
research, we screened several cell lines from different species. As
shown in Fig. 2a. All tested cells infected with the pseudotyped EBOV
were found to have relatively high RLU values, except for C6/36 cells.
These findings suggested that the rescued pseudotyped EBOV could infect
different target cells in this research, except C3/36 cells from
mosquitoes, which coincides with the characteristics of EBOV (EBOV has
no infectivity in C6/36 cells), indicating that the infective ability of
pseudotyped EBOV was higher in Huh-7 cells than in any other cell lines,
as a result, Huh-7 cells were selected as a tool to study kinetics of
pseudotyped EBOV production.
The RLU values of pseudotyped EBOV rescued at different time points are
shown in Fig. 2b, pseudotyped EBOV could reach a peak RLU value at 24 h
post-transfection. The titers of pseudotyped EBOV that were rescued at
different times are shown in Fig. 2c, the pseudotyped EBOV had the
highest titer when rescued between 24-36 h post- transfection. To
confirm the single-cycle infectivity of the pseudotyped EBOV, we
harvested the supernatant from Huh-7 cells infected with pseudotyped
EBOV, which was used to reinfect Huh-7 cells. Two days later, RLU
activity and titer of the supernatant were detected, representing the
second-cycle characteristics of pseudotyped EBOV. As shown in Fig. 2d
and 2e, the RLU activity of the second life cycle was substantially
lower than that of the first life cycle, and the titer of pseudotyped
EBOV in its second life cycle could not be detected. These findings
showed that there was only one life cycle of pseudotyped EBOV, implying
that this pseudotyped EBOV should be safe for research.