Construction and characterization of a stable subgenomic Dengue virus type 2 replicon system for antiviral compound and siRNA screening

Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2'-C-methyladenosine (EC50 = 2.42 +/- 0.59 [mu]M), or ribavirin (EC50 = 6.77 +/- 1.33 [mu]M), mycophenolic acid (EC50 = 1.31 +/- 0.27 [mu]M) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery.