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.

Infectious virus from transfected and infected 293T cells was quantified by endpoint dilution assay on Vero cells (TCID 50 ) by measuring ZIKV-associated cytopathic effect (CPE). As observed above, 293T cells transfected with the Pol(Ϫ) MR766 plasmid failed to release detectable levels of infectious virus (data not shown). At 24 h after transfection with the wild-type MR766 plasmid, 293T cells produced 1.6 ϫ 10 4 TCID 50 /ml, which increased to 2.4 ϫ 10 7 and 2.5 ϫ 10 8 TCID 50 /ml at 2 and 3 days posttransfection, respectively (Fig. 4B ). Parallel infection of 293T cells with the parent MR766 virus resulted in supernatants containing between 2.6 ϫ 10 7 and 2.2 ϫ 10 8 TCID 50 /ml. However, it should be noted that the 24 h time point likely contains residual input virus, as we chose to conduct overnight infections and did not wash input virus away prior to collecting the first time point. Nevertheless, comparing the kinetics of virus released following plasmid transfection with that following virus infection may not be relevant because RNA delivered in a virion can be directly translated and replicated while plasmid-expressed RNA requires transcription, splicing, and export from the nucleus.