Fig. 1

Schematic of the strategy used to create the chimeric genome of ZIKV/THOV(prM-E). A Construction of pUC19-F1a. Initial experiments required the use of two plasmids, designated pUC19-F1 and pUC19-THOVprME. pUC19-F1 contains an insect virus promoter (OpIE2-CA), followed by ZIKV sequence (ZIKV genomic position 1-3460) that spans all of the 5′ UTR through to the first 971 nt. of the NS1 gene. pUC19-THOVprME contains the prM-E gene sequences of T’Ho virus (T’Ho virus genomic position 455-2461). The aforementioned plasmids were used as templates in PCRs. One PCR amplified all of pUC19-F1, except for ZIKV prM-E. The reaction was performed using a forward chimeric primer specific to the 3′ and 5′ ends of the T’Ho virus E gene and ZIKV NS1 gene, respectively, and a reverse chimeric primer specific to the 3′ and 5′ ends of the ZIKV C gene and T’Ho prM gene, respectively. Another PCR was designed to amplify all of T’Ho virus prM-E from pUC19-THOVprME. The reaction was performed using a forward chimeric primer specific to the 3′ and 5′ ends of the ZIKV C gene and T’Ho virus prM gene, respectively and a reverse chimeric primer specific to the 3′ and 5′ ends of the T’Ho virus E gene and ZIKV NS1 gene, respectively. The two amplicons were joined by Gibson assembly, yielding a plasmid designated as pUC19-F1a. The newly created plasmid contains the prM-E sequences of T’Ho virus, flanked at the 5′ end by OpIE2-CA and the 5′ UTR and C sequences of ZIKV and flanked at the 3′ end by the first 971 nt. of the NS1 gene of ZIKV. B Generation of the full-length chimeric orthoflavivirus genome. Subsequent experiments required the use of pUC19-F1a and two additional plasmids, designated as pUC19-F2 and pUC19-F3. pUC19-F2 contains ZIKV sequence that spans the last 138 nt. of the NS1 gene through to the first 402 nt. of the NS5 gene (ZIKV genomic position 3413-8071). pUC19-F3 contains ZIKV sequence that spans all of the NS5 gene, except for the first 344 nt., and all of the 3′ UTR (ZIKV genomic position 8016-10,807), followed by the hepatitis delta virus anti-genomic ribozyme sequence (HDVr) and simian virus 40 polyadenylation signal (SV40p). The aforementioned plasmids were used as templates in PCRs that amplified all of the viral sequences and none of the cloning vector (pUC19) sequences. Primers were designed so that each amplicon contained an overlap of about 50 bp with the adjacent amplicon(s). The three amplicons were joined by Gibson assembly, yielding a linear chimeric orthoflavivirus genome flanked by OpIE2-CA sequence at its 5′ end and HDVr and SV40p sequences at its 3′ end. Sequences are color-coded: pUC19 (black), OpIE2-CA (green), ZIKV (red), T’Ho virus (blue) and HDVr/SV40p (orange). PCR primers are denoted as small arrows, which are color-coded according to the sequence to which they bind. Chimeric primers are represented by bicolored arrows