Zika and Dengue Virus Infection in RPE Cells

Human primary retinal pigmented epithelial (RPE) cells (Lonza, Walkersville, MD) were cultured in RtEGM^™Bulletkit^® media as per manufacturer’s instruction (Lonza). Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were cultured in DMEM, and EMEM media supplemented with 10% FBS, 10 µg/ml L-glutamine, and 1% penicillin and streptomycin solution as per manufacturer’s recommendation. ZIKV Virus (ZIKV) strain PRVABC59, NR-50240, originally isolated from human blood in Puerto Rico in December 2015, and DENV type 2 strain NR12216 were obtained through BEI Resources, NIAID, NIH. This ZIKV strain is 97–100% genetically similar to the current ZIKV strain circulating in Brazil^{57 (link)}. ZIKV and DENV were propagated in ATCC CCL-81 Vero cells and ATCC CRL-1660 C6/36 cells respectively, and titers were determined by plaque assay. RPE cells were infected with ZIKV and DENV at MOI of 1 for the indicated time points as described previously⁷ . For ABCG1 functional studies, Pr. RPE cells were treated with a pharmacological inhibitor of ABCG1, Benzamil (50 µM) (Tocris Biosciences, Minneapolis, MN), and cholesterol-water soluble (10 µM) (Sigma Aldrich, St Louis, MO) 1 h before ZIKV challenge.