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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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Comparison of the replication and infectivity of ZL1(V2634M) with wild type ZL1 in Vero E6 cells. (a) Replication analysis of wild type ZL1 with mutated ZL1(V2634M). The ZIKV subgenomic replicon with a Renilla luciferase (SGR, top) was engineered with mutation (V2634M). Equal amounts of ZL1 and ZL1(V2634M) replicon RNA (5ug) were electroporated into Vero E6 cells. Luciferase signals were measured at the indicated time points. A defective replicon containing an inactive NS5 polymerase with GDD-to-AAA mutation was included as a negative control. The averages of three replicates are presented. Error bars represent standard deviations from the means. (b/c) Comparison of the replication and virus production by wild type ZL1 and ZL1 (V2634M). Vero E6 cells were infected with ZL1 or ZL1(V2634M) at MOI = 0.01, the cell pellets and supernatants were collected at 6,24,48,72,96,120 hours post infection. Intracellular ZIKV RNA copies and titer of cell culture supernatant were detected by q-RT-PCR and plaque assay, respectively. Data represent the means of three independent assays; Error bars represent standard deviations from the means. (d) Western blot analysis of viral proteins in infected cells. The cell pellets were collected at different time points following infection (MOI = 0.01), and the levels of ZIKV protein (Capsid, E, NS1, NS3, NS5), STAT2 and Tubulin were analysed by western blot. (e) Nuclear localization of viral NS5 protein in Vero E6 cells. ZL1 and ZL1(V2634M) virus infected cells were fixed and stained with homemade Rabbit anti-NS5 antibody at 48 h or 96 h post electroporation. Green and blue represent NS5 and nucleus (stained with DAPI), respectively.