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.

Comparison of the growth properties of rescued and parental MR766 viruses. The previously developed systems for producing infectious 2010 Cambodia ZIKV and 2015 Brazil ZIKV from plasmids showed that the rescued viruses were attenuated in comparison to the parental inoculum (5, 8) . To compare the fitness levels of our parental and rescued MR766 viruses, we first calculated their Vero cell specific infectivities. To ensure that only virion-associated RNA was quantified, the rescued virus was initially passaged twice at an MOI of 0.1 in Vero cells. The rescued and parental MR766 viruses exhibited similar and not significantly different specific infectivity values of 218 and 130 RNA molecules per TCID 50 (Fig. 4C ). To directly compare growth properties of the 293T cell-rescued MR766 virus to those of the parental inoculum using a strategy similar to that used on previously published systems, we performed plaque assays ( Fig. 4D and E) . When visualized at 4 days postinfection, the rescued and parental MR766 ZIKV-induced plaques exhibited a 1.35-fold, but not statistically significant, difference. According to the work of Tsetsarkin et al., the rescued 2015 Brazil ZIKV isolate made plaques that were 2.5-fold smaller than the parental virus plaques at this time point (8) . A retrospective analysis of plaque sizes in the publication by Shan et al. showed that their rescued 2010 Cambodia ZIKV produced plaques that were approximately 3.3-fold smaller than parental virus plaques at 4 days postinfection (5) . However, we were able to detect slight attenuation of our rescued virus, as we observed statistically significant 2.4-and 2-fold differences in rescued and parental MR766 ZIKV plaque sizes when we stained our plaque assays at 5 and 6 days postinfection, respectively ( Fig. 4D and E) .