Fig. 2

Mosquito cells support the replication of ZIKV/THOV(prM-E). A Western blot analysis of C6/36 cells inoculated with ZIKV/THOV(prM-E). C6/36 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 C6/36 cells prior to the experiment. Lysates were harvested at 5 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. The arrows show the expected migration positions of ZIKV C and NS1 (molecular weights: 12, 19 and 48 KDa, respectively) and cellular β-actin (molecular weight: 42 KDa). B IFA analysis of C6/36 cells inoculated with ZIKV/THOV(prM-E). C6/36 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 C6/36 cells prior to the experiment. Cells were fixed with methanol at 5 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 3,500X was used. Scale: 100 µM