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. 2020 Oct 14;16(10):e1008951. doi: 10.1371/journal.ppat.1008951

Fig 1. Generation of a ZIKV full-length reverse genetics system.

Fig 1

A low-copy number plasmid (pACYC177) was modified between the XhoI and ClaI restriction sites to include a 490-bp fragment containing the T7 promoter sequence, unique restriction sites and the HDV ribozyme sequence. cDNA fragments spanning the entire ZIKV genome were synthesized from stock viral RNA and cloned into the modified plasmid. Two separate constructs were utilized to manage the toxicity of the NS1 region: pZika(Hond)-ABD and pZika(Hond)-NS1. Plasmid pZika(Hond)-ABD encompasses the entire ZIKV genome, but lacks the majority of the NS1 region, which is replaced with a NotI restriction site. Plasmid pZika(Hond)-NS1 contains a 965-bp fragment of the NS1 region flanked with EcoRI restriction sites. To distinguish between rescued and parental virus, genetic markers were placed into the cDNA clone within the E gene to eliminate the AvrII restriction site while simultaneously generating a BamHI restriction site.