2.4 Insect cell culture virus growth curves
The insect cell lines used for this study were Ct cells, derived fromCx. tarsalis embryos (Centers for Disease Control and Prevention)(Chao & Ball, 1976), Aag2 Ae. aegypti high passage cells, also derived from embryos(Chao & Ball, 1976), and ATC10 (CCL-125 (ATCC)), a larval-derived cell line(Singh, 1971). Virus strains RVFV-ZH501, the parental wild-type to DDVax(Bird et al., 2008), MP-12(Turell & Rossi, 1991) and DDVax were subjected to growth curves in mosquito cell culture (ATC-10, Aag2, CT) using Schneider’s media (10% FBS (or 20% FBS for ATC-10s), 1% non-essential amino acids, 1% L-glutamine, 1% penicillin/streptomycin). An MOI of 0.01 was used for all infections. Aliquots were removed at daily timepoints for 1-6 days post-infection (dpi). At each timepoint, 400 µl cell culture supernatant was removed, and media was replaced into the T-flask. Aliquots were supplemented with 20% FBS as a cryoprotectant and stored at -80°C until titrations were performed.