Fig. 2

In vitro characterization of FPzenv-mediated transgene expression and VLP formation. a Expression of the Env protein of ZIKV by the FP recombinants in Vero cells. Vero cells were infected by the FP recombinants and examined using Western blotting, to determine the Env protein expression. The Env protein was always detected both when using the monoclonal or the polyclonal antibodies after infection with either ZIKV (Z, lanes 2) or FPzenv (zenv, lanes 4). Mock infected cells (m, lanes 1) and cells infected with FP wild-type (wt, lanes 3) were used as negative controls. b Heterologous protein expression by immunofluorescence in the CEFs and the Vero and MRC-5 cells. Immunofluorescence of the infected cells was performed to determine the subcellular localization of the Env protein expressed by FPzenv. The Env protein was expressed mainly in the cytoplasm (2a-2b-2c), and the intensity of the fluorescence signals was generally lower in cells infected with the recombinant than in the same cells infected with ZIKV (3b-3c). ZIKV did not infect the CEFs (3a). No immunofluorescence was detected in the FP-wild-type-infected cells used as negative controls (1a-1b-1c). c Expression of the env transcripts over time by FPzenv in replication-restrictive Vero cells. After infection of the Vero cells with FPzenv, the expression of the transgene was evaluated by RT-PCR every 3 days, over 27 days. The expression levels for FPzenv transcripts (661 bp) remained up to day 18 p.i.. Amplification of β-actin mRNA (518 bp) is shown. d Electron microscopy. Vero cells were infected with FPzenv to verify production of virus-like particles (VLPs). Left. Some empty VLPs were seen (white arrows), as well as clusters of FPzenv recombinants corresponding to the viral inoculum (black arrows) and DNA viral factories (V); bar, 0.2 µm. Inset, VLPs enlargement; bar, 50 nm. Right. ZIKV-infected cells (black arrows) were used as the positive control, and clusters of virus particles (50 nm, black arrows) were seen inside the cytoplasm; bar, 0.2 µm