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. 2018 Nov 26;8:17385. doi: 10.1038/s41598-018-35724-6

Figure 1.

Figure 1

Generation and in vitro characterization of MVA-ZIKV. (a) Scheme of the MVA-ZIKV genome map. The ZIKV signal peptide (sp) following by the ZIKV prM-E structural genes (isolate Z1106033) are driven by the novel VACV synthetic pLEO160 promoter and are inserted within the VACV TK viral locus (J2R). The deleted VACV C6L, K7R, and A46R genes are indicated. TK-L, TK left; TK-R, TK right. (b) PCR analysis of the VACV TK locus. Viral DNA was extracted from DF-1 cells mock infected or infected at 5 PFU/cell with MVA-ZIKV, MVA-Δ-GFP, or MVA-WT. Primers spanning the TK locus-flanking regions were used for PCR analysis of the ZIKV genes inserted within the TK locus. DNA products are indicated by an arrow on the right. A molecular size marker (1-kb ladder) with the corresponding sizes (base pairs) is indicated on the left. (c) Expression of ZIKV prM and E proteins. DF-1 cells were mock infected or infected at 5 PFU/cell with MVA-ZIKV, MVA-Δ-GFP, or MVA-WT. At 24 hpi, cells were lysed, fractionated by 10% SDS-PAGE, and analyzed by Western blotting. Arrows on the right indicate the positions of the ZIKV prM and E proteins, the VACV E3 protein or β-actin. The sizes of standards (in kDa) are indicated on the left. (d) Viral growth kinetics of MVA-ZIKV. Monolayers of permissive DF-1 or non-permissive HeLa cells were infected at 0.01 PFU/cell with MVA-WT or MVA-ZIKV. At different times postinfection (0, 24, 48, and 72 hpi), virus titers in cell lysates were quantified by a plaque immunostaining assay. The means of results from two independent experiments are shown. (e,f) Stability of MVA-ZIKV. MVA-ZIKV (P2 stock) was continuously grown in DF-1 cells to passage 9 (e) and at passage 9, 24 individual plaques were picked (f). Virus stocks from each passage and from the 24 individual plaques were used to infect cells and the expression of ZIKV prM and E proteins was determined by Western blotting. Rabbit anti-VACV E3 protein antibody was used as a VACV loading control. Arrows on the right indicate the position of the ZIKV prM and E proteins, and the VACV E3 protein. The sizes of standards (in kDa) are indicated on the left.