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. 2018 Mar 26;9:1230. doi: 10.1038/s41467-018-03662-6

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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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Fig. 1 — SCV-ZIKA/CHIK construction, rationale and characterization. a VACV-CHIK was generated from vaccinia virus (VACV) by insertion of the CHIKV structural protein expression cassette inserted into the A39R locus. SCV-ZIKA/CHIK was constructed from VACV-CHIK by insertion of ZIKV prME expression cassette into the B7R-B8R locus and concurrent deletion of D13L. b After vaccination the genome is amplified in SCV-ZIKA/CHIK-infected host cells and CHIKV and ZIKV immunogens are expressed from the amplified genomes. Due to the targeted deletion of D13L, no viral progeny are generated. c PCR of SCV-ZIKA/CHIK and VACV infected SCS cells confirming insertion of CHIKV and ZIKV genes into A39R and B7R-B8R loci, respectively, and deletion of D13L. d Immunoblot of SCV-ZIKA/CHIK and SCV-cont infected SCS and HeLa cells using an anti-ZIKV E antibody, with recombinant ZIKV E (rE) as a positive control. e Immunoblot of SCV-ZIKA/CHIK and SCV-cont infected SCS and HeLa cells using a polyclonal anti-CHIKV mouse anti-serum. f Lysates of SCV-ZIKA/CHIK-infected cells after one (P1) and ten passages (P10) in SCS cells, were analyzed by PCR (as in c) for retention of inserts in the A39R and B7R-B8R loci. g Quantitative PCR of ZIKV M DNA of lysates described in f, normalized to VACV G1L DNA. Error bars represent standard deviation (n = 4)