Mutational analyses of the predicted stem-loop (SL) structure in the junction region of the HEV genome. (A) Mutations were introduced into the stems and loop sequences of the SL structure in mutants M1 to M9, M59, and M78 for the HEV Rluc replicon. (B) Relative luciferase activities in Huh7 S10-3 cells transfected with HEV Rluc mutants M1 to M9, M59 (i.e., M5 plus M9), M78 (i.e., M7 plus M8), HEV Rluc MGAA (negative control), and the wild-type Rluc replicon (HEV-RLuc). The relative luciferase activities are shown at 5 days posttransfection and normalized with cotransfected firefly luciferase RNA. Data are from an average of eight separate replicate experiments, and the error bars indicate standard deviations (SD). The differences in signal produced by HEV Rluc mutants and the wild-type Rluc replicon were compared by one-way analysis of variance (ANOVA) using the Kruskal-Wallis test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; RLU, relative light units.