The Zika virus infectious clone was used as a template for the generation of DVG-encoding clones, using primers to excise deleted region of interest. The deleted nucleotides in each DVG clone were: 581-3250 for DVG-A, 339-9033 for DVG-B, and 5351-5668 for DVG-C. Zika virus replicons were generated as previously described12. Briefly, the region encoding the structural proteins (C, Pr, M, and E) was replaced by a cassette containing C38-NanoLuc-2A-E30 (the N-terminal 38 amino acids of C protein, NanoLuc reporter, foot-and-mouth disease virus 2 A protease, and the C-terminal 30 amino acids of the E protein). The N-terminal 38 amino acids of the C protein contains the cis-acting element of nucleotides complementary to the 3’ cyclization sequence57. The C-terminal 30 amino acids of E correspond to the transmembrane domain required to maintain the topology of NS1 in the ER. In addition, the intron present in NS1 in the Zika virus clone was removed. A mutant replicon plasmid was generated for use as a negative control, by replacing the catalytic motif G664D665D666 in the viral polymerase NS5 by three alanine residues (inactive replicon). The DVG-A reporter clone was generated similarly; with NanoLuc-2A introduced at the deletion site, namely between the N-terminal 38 amino acids of Pr and the C-terminal 98 amino acids of NS1.

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