Investigating JEV Replicon Regulation by CW-33A

A JEV-EGFP replicon, previously constructed in our laboratory^{10 (link)}, was used to explore whether CW-33A influenced viral protein translation and RNA genome replication stages of JEV life cycle. TE-671 cells were transfected with JEV-EGFP replicon and simultaneously treated with 10 μM of CW-33A or CW-33. After 36 hours post treatment, the images of replicon-derived EGFP reporter in JEV-EGFP replicon-transfected cells were taken by fluorescent and optical microscopies. The fluorescent intensity of EGFP reporter in treated transfected cells was quantified by Image J. For detecting the synthesis of JEV sense and antisense genomes, total RNAs of replicon-transfected TE-671 cells treated with CW-33A or CW-33 were purified using PureLink Mini Total RNA Purification Kit (ThermoFisher), and performed using SYBR Green-based real time PCR with JEV-specific primer pairs, as described in our prior report [9]. The corresponding threshold cycle value(C_T) for each sample was measured by 7300 Realtime PCR system (Applied Biosystems). Relative levels of JEV sense and antisense genomes were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then quantitated.