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.

Virus produced from the previously published 2010 Cambodia and 2015 Brazil ZIKV isolate rescue systems exhibited slower growth kinetics than the parental viruses, potentially due to sequence diversity within the parental virus populations that provided a fitness advantage (5, 8) . Conversely, our rescued MR766 virus grew identically to the parental isolate in Vero cell multicycle growth curve experiments and produced similarly sized plaques in standard 4-day infection plaque assays. Thus, even though we observed plaque size differences between our parental and rescued virus in extended infection, and thus likely more sensitive plaque assays, we believe that our MR766 rescued virus is less attenuated than the rescued 2010 Cambodia and 2015 Brazil ZIKV. It is unlikely that the methods of rescue (transfection of in vitro transcribed RNA versus plasmid DNA) impacted virus attenuation. Instead, we hypothesize that viral genetics influenced postrescue attenuation, and this may be greatly influenced by the passage history of a viral isolate. The 1947 MR766 isolate may not require the same sequence diversity as the 2010 Cambodia and 2015 Brazil ZIKV isolates to reach optimal fitness in Vero cells. The MR766 virus has been subjected to more rounds of amplification in Vero cells than these more recent strains, which may have preadapted it to efficient cell culture replication. Ultimately, rescued virus attenuation should not be a serious complication to experiments to study viral determinants of replication or pathogenesis. Researchers using any ZIKV rescue system would merely need to monitor the stability of engineered sequences of interest and compare phenotypes between wild-type and mutant viruses rescued in parallel.