ZIKV virus isolate Brazil-ZKV2015 (accession number KU497555) was used to design NS1 transgenes, which were produced synthetically and codon optimized for enhanced mammalian expression by GeneArt (Germany). ZIKV NS1 was cloned into pVax (Invitrogen) downstream of the CMV promoter, and a Kozak translation initiation sequence was included. Three plasmids were generated encoding either wild-type NS1 viz. (i) pVAX-NS1, or secreted NS1 generated by the upstream introduction of the human TPA leader sequence, viz. (ii) pVAX-tpaNS1 (63). To generate secreted NS1 fused to the oligomerization domain of the C4b-p (pVAX-tpaNS1-IMX313), TPA and the oligomerization domain of C4b-p (IMX313) were introduced at the N and C termini, respectively, of the ZIKV NS1 gene as we described (35, 64). Plasmids were produced with QIAGEN endotoxin-free gigaprep kits. Sequences were confirmed by double-stranded sequencing.

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