Figure 5.

Trans complementation of class III and V mutants. (A) IFA of BHK-NS2A-HA cells. The BHK-NS2A-HA cells constitutively express WT ZIKV NS2A fused with a C-terminal HA-tag. The cells were fixed with paraformaldehyde and stained with an HA-tag antibody. (B) Western blot of BHK-NS2A-HA cells. The expression of WT NS2A-HA from BHK-NS2A-HA cells was detected using an HA-tag antibody. Both passage one (P1) and 20 (P20) BHK-NS2A-HA cells were analyzed. Naïve BHK-21 cells were included as a negative control. (C) WT and class III and V genomic RNAs were electroporated into BHK-NS2A-HA cells. IFA was performed on the transfected cells to detect viral E protein expression. (D) Viruses rescued from the BHK-NS2A-HA cells (harvested at 96 h p.t.) were used to infect naïve Vero cells. The infected Vero cells were subjected to IFA for E protein expression. (E) Extracellular viruses were harvested from the supernatants of RNA-transfected BHK-NS2A-HA cells. The harvested viruses were serially diluted to infect naïve Vero cells in 96-well plates. At 24 h p.i., IFA was performed to quantify the viral titers by counting the number of E-positive cells. The results show averages and standard deviations from triplicate samples. Viral RNAs in the supernatants of transfected BHK-NS2A-HA cells were isolated at 96 h p.t. and subjected to RT-PCR. Positive RT-PCR products were sequenced to confirm the engineered mutations and to identify potential second site change(s). (F) The supernatants of infected Vero cells from (D) were collected at 72 h p.i. to infect naïve Vero cells for a second round. IFA was performed on these cells for viral E-positive cells.