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.

Plaque assays. One day prior to infection, 7.5 ϫ 10 5 Vero cells were seeded in polylysine-coated 12-well plates in DMEM with 2% FBS. For infection, medium was replaced with 300 l of 10-fold serial dilutions (with a starting MOI of 5) of either the parental or rescued virus. After 2 h at 37°C, 1.5 ml 0.8% methylcellulose in DMEM with 2% FBS was added to each well. At 4 to 6 days postinfection, cells were fixed in 1 ml 4% paraformaldehyde for 1 h at room temperature. Cells were washed twice with PBS and stained with 500 l of 0.5% crystal violet solution in ethanol. Plates were washed twice with water and air dried. Plates were scanned, and plaque sizes were measured using ImageJ software (National Institutes of Health, Bethesda, MD; http://imagej.nih.gov/ij/). Statistical analysis. Data were analyzed using an unpaired Student t test (*, P Ͻ 0.05; **, P Ͻ 0.005; ***, P Ͻ 0.0005) on Prism software (GraphPad Software). Values in graphs represent the mean and standard error from experiments performed in triplicate or quadruplicate, with between two and five independent experiments.