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.

Flavivirus sequences are notoriously difficult to propagate in bacteria, likely because the presence of cryptic bacterial promoters permits expression of viral proteins that result in bacterial toxicity (reviewed in references 9 and 10). Numerous strategies have been devised to limit and prevent bacterial death, including the use of bacteria that are more resistant to this toxicity, very-low-copy-number plasmids such that fewer viral translation products are produced, and alternative organisms such as yeast for plasmid propagation. Another strategy entails the separation of the viral genome into multiple plasmids, where each piece can be excised by restriction enzyme digestion and ligated into the full-length viral cDNA, which can then serve as the template for in vitro transcription. Here, we used an alternative strategy to stabilize the 1947 MR766 ZIKV genome (2) in bacteria that has been successfully used to stabilize other positive-sense RNA viruses, including transmissible gastroenteritis coronavirus and Japanese encephalitis virus (11, 12) and, more recently, the 2015 Brazil ZIKV genome (8) . We identified the major region of the MR766 genome that induced toxicity in bacteria and then cloned this sequence with a synthetic intron insertion to interrupt viral translation in bacteria. This RNA was spliced in mammalian cells to recreate the authentic viral genome, which efficiently initiated infectious virus production.