Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone

Schwarz, Megan C.; Sourisseau, Marion; Espino, Michael M.; Gray, Essanna S.; Chambers, Matthew T.; Tortorella, Domenico; Evans, Matthew J.

In this work, we describe the creation of a plasmid-based rescue system for the prototype Uganda 1947 MR766 ZIKV. Other groups have published ZIKV infectious virus rescue systems. The first such system was a clone of the 2010 Cambodia ZIKV isolate cDNA (5) . Here, a T7 RNA polymerase promoter directed in vitro transcription of the viral RNA for subsequent transfection into permissive host cells for initiation of infection. Our approach to express the ZIKV RNA from an RNA polymerase II promoter with a ribozyme positioned at the 3= end to trim the viral transcript allowed us to avoid the potentially arduous in vitro transcription step and directly transfect the plasmid into host cells. The Shan et al. system lessened the toxicity of ZIKV cDNA in bacteria by using a very-low-copy-number bacterial plasmid. In our system, the deleterious sequence within the genome was disrupted by the addition of an intron, which allowed the successful amplification of this sequence in a high-copy-number plasmid (Fig. 1) . In a more recent publication, Tsetsarkin et al. described their derivation of a similar plasmidbased rescue system for a 2015 Brazil ZIKV isolate (8) . They also found that the toxicity of the ZIKV cDNA could be reduced by insertion of introns in the viral open reading frame.