Preventative Vaccines for Zika Virus Outbreak: Preliminary Evaluation

Kim, Eun; Erdos, Geza; Huang, Shaohua; Kenniston, Thomas; Falo, Louis D.; Gambotto, Andrea

ZIKV stocks were provided by Dr. Rober Tesh of University of Texas Medical Branch. Vero cells were infected with ZIKV DAKAR41542 at MOI of 0.01 and incubated until the monolayer showed significant cytopathic effect. Culture supernantant was clarified by centrifugation at 3000g for 15 min. Virus was precipitated overnight by addition of NaCl (0.4 M) and 6% polyethylene glycol. After centrifugation at 10,000g for 30 min, the viral pellet was re-dissolved to 1/100 of the original volume in PBS and centrifuged on a 5 to 50% sucrose gradient at 90,000g for 3 h, followed by dialysis with PBS buffer. The virus was diluted to a proper concentration with 5% Trehalose Buffer (20 mM Tris, pH 7.8, 75 mM NaCl, 2 mM MgCl 2 , 5% Trehalose, 0.0025% Tween 80) and kept at −80°C. For the virus titer, vero cells were seeded in a sixwell plate at 1 × 10 5 cells per well. The next day, cells were infected with log dilutions of ZIKV for 1 h and overlayed with 1% methyl cellulose media containing 5% fetal bovine serum. After three days of infection, cells were stained with 1% crystal violet. Plaques were counted and titers were calculated by multiplying the number of plaques by the dilution and dividing by the infection volume.