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. 2018 Jul 12;8:10491. doi: 10.1038/s41598-018-28890-0

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Construction of ZIKV infectious clone and virus rescue (a) Schematic diagram of full-length ZIKV infectious cDNA clone. 17 nucleotides of ZIKV sequence were mutated without altering the amino acid sequence to eliminate the activity of predicted bacterial promoters. Red bars in the box represent engineered mutation sites of ZIKV genome (Genbank: KU321639.1). (b) Analysis of linearized ZIKV infectious clone pZL1 on a 1% agarose gel. (c) Analysis of in vitro transcript from pZL1 on a 1% agarose gel. (d) Indirect immunofluorescence analysis of viral protein expression in Vero E6 cells after electroporation with full-length ZL1 RNA. Vero E6 cells were electroporated with 5ug of full-length ZL1 RNA. Cells were fixed and stained the viral E protein by using flavivirus antibody (4G2) at 5 days post electroporation. Green and blue represent E protein and nucleus (stained with DAPI), respectively. (e) Cytopathic effect (CPE) on Vero E6 cells at day 4 post infection. Fixed cells were stained by 0.05% crystal violet solution. (f) Growth kinetics of rescued ZL1 (electroporation), ZL1 (re-infection, MOI = 0.01), isolated strain SZ-WIV01 (infection MOI = 0.01) viruses in Vero E6 cells, or ZL1 (re-infection, MOI = 0.01) virus in IFN competent cell A549. Data represent the means of three independent assays; Error bars represent standard deviations from the means. (g) Western blotting analysis of expression of rescued ZL1 proteins. Vero E6 cells were infected with ZL1 virus (MOI = 0.01). Viral proteins were detected by using home-made polyclonal antibodies at 3 days post infection.