Figure 1. Lentiviral Delivery of FvE ^J shRNA Suppresses JEV Replication in Neuro 2a Cell Line

(A) Northern blot shows intracellular processing of FvE^J shRNA. RNA extracted from Neuro 2a cells stably transduced with shFvE^J or shLuc lentivirus was probed with³²P end-labeled synthetic FvE^J siRNA sense strand to detect intracellular processing of shRNA. Antisense strand of the synthetic FvE^J siRNA (siFvE^J) was used as positive control. Before loading, the samples were normalized for total RNA content.

(B) shFvE^J inhibits JEV replication in Neuro 2a cells. Mock- or lentivirally transduced Neuro 2a cells were challenged with JEV at a MOI of 1, and the viral replication monitored 60 h later by flow cytometry after staining the cells with a JEV envelope-specific antibody. Percent of infected cells is indicated. The results are representative of at least three independent experiments.

(C) Titration of shFvE^J-induced inhibition of JEV replication. Neuro 2a cells transduced with shFvE^J or control shLuc lentivirus were challenged with the indicated MOIs of JEV, and viral replication was assessed by flow cytometry at different times postinfection. Percent inhibition of viral replication compared to mock-transduced cells is shown. Results are representative of three experiments.

(D) shFvE^J inhibits accumulation of JEV genomic RNA. Total RNA obtained from the control shLuc- or shFvE^J lentivirus-transduced cells, which were either uninfected (UI) or infected with JEV, was probed with JEV- or β-actin cDNA in a Northern blot analysis.