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.

To characterize the ability of the above plasmids to produce ZIKV proteins in mammalian cells, we transfected 293T cells with the wild-type and Pol(Ϫ) MR766 ZIKV plasmids and infected cells in parallel with the parental MR766 inoculum at an approximate multiplicity of infection (MOI) of 0.03 50% tissue culture infective dose (TCID 50 ) per cell. Three days later, the cells were fixed and immunostained with antibodies against the ZIKV E and NS3 proteins ( Fig. 2A) . While all transfected and infected cells expressed both viral proteins, cells transfected with the wild-type MR766 plasmid exhibited larger amounts of antibody staining than those transfected with the Pol(Ϫ) plasmid. This difference was confirmed by quantifying E protein immunostaining by fluorescence-activated cell sorting (FACS) analysis (Fig. 2B ). To examine viral protein expression over time, cells collected at various time points following transfection or infection were analyzed by FACS for E protein antibody staining or by immunoblotting of cell lysates with NS3 antibodies ( Fig. 2C and D, respectively). While the percentage of E protein-positive cells increased over 3 days following transfection with the wild-type MR766 plasmid and infection with the parental MR766 virus, the E protein-positive Pol(Ϫ) plasmid-transfected cells remained below 5% throughout this time course (Fig. 2C ). NS3 protein levels also increased over time in both wild-type plasmid-transfected and parental virus-infected cell populations and yet remained below the limit of detection in Pol(Ϫ) plasmid transfected cells. These results confirmed that although both the wild-type and Pol(Ϫ) MR766 ZIKV plasmids were able to express viral proteins, protein levels were higher and were detected in a greater percentage of cells transfected with the wild-type plasmid, which may be due to viral RNA replication and viral spread.