Fig. 3
Vertebrate cells support the replication of ZIKV/THOV(prM-E). A Western blot analysis of Vero cells inoculated with ZIKV/THOV(prM-E). Vero cells approaching confluency in 25 cm2 culture flasks were inoculated with ZIKV/THOV(prM-E) or ZIKV at a multiplicity of infection of 0.1 or they were inoculated with media only (lane 1–3, respectively). The chimeric virus had undergone two consecutive passages in Vero cells prior to the experiment. Lysates were harvested at 3 days p.i. then equal amounts of protein were resolved on 8–16% Tris–glycine gels and analyzed by Western blot using (i) anti-ZIKV C polyclonal antibody, (ii) anti-ZIKV prM polyclonal antibody, (iii) anti-ZIKV NS1 polyclonal antibody or iv) anti-β-actin polyclonal antibody to ensure there was approximately equal loading in each lane. The arrows show the expected migration positions of ZIKV C, prM, and NS1 (molecular weights: 12, 19 and 48 KDa, respectively) and cellular β-actin (molecular weight: 42 KDa). B IFA analysis of Vero cells inoculated with ZIKV/THOV(prM-E). Vero cells approaching confluency in 35 mm2-well culture dishes were inoculated with ZIKV/THOV(prM-E), ZIKV or media only (rows 1–3, respectively). The chimeric virus had undergone two consecutive passages in Vero cells prior to the experiment. Cells were fixed with methanol at 3 days p.i. and immunostained with an anti-ZIKV C polyclonal antibody (column 2) or a pooled suspension of heterologous hyperimmune polyclonal antibodies to ZIKV and several other orthoflaviviruses (column 3), followed by a pooled suspension of Alexa Fluor 594-conjugated donkey anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG. DAPI was used to visualize the nucleic (column 1). Merged images are also shown (column 4). A magnification of 3500X was used. Scale: 100 µM. C Comparison of the plaque morphologies of ZIKV/THOV(prM-E) and ZIKV. Confluent monolayers of Vero cells in 35 mm2 culture dishes were inoculated with (A) ZIKV/THOV(prM-E) or (B) ZIKV then incubated for 5 days and fixed. Two replicate experiments were performed and at least 40 plaques were measured for each virus