RHDV VPg protein is essential for RHDV translation.

(A) Schematic diagram of the pRHDV-luc, pRHDV-luc/ΔVPg, and pRHDV plasmids [16]. The coding regions of viral structural proteins were replaced with Fluc using fusion PCR. In the fusion PCR results, a gray box denotes the coding region of the viral structural proteins, and lines indicate the 5′ and 3′ UTRs. (B) Thirty-two hours after transfection, the luciferase mRNA levels in cells transfected with pRHDV-luc, pRHDV-luc/ΔVPg + pVPg, or pRHDV-luc/ΔVPg were evaluated by qRT-PCR. The Student’s t test and ANOVA were applied for the statistical analyses, P <0.05 was considered as significantly different (*), and P <0.01 was considered as extremely significant different (**). (C) The expression level of trans-supplemented VPg was evaluated by immunoblotting with VPg polyclonal antibodies. Line 1: pVPg; line 2: pcDNA3.1 vector; line 3: blank control. (D) Relative luciferase activity in RK13 cells carrying pRHDV-luc/ΔVPg, trans-supplemented pVPg, and the parental genotype pRHDV-luc at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-transfection. The luciferase activity in RK13 cells was evaluated by measuring the firefly luciferase activity at different time points after transfection. Renilla luciferase activity measured at the same time points was used to normalize the transfection efficiency. The differences in luciferase activities associated with pRHDV-luc/ΔVPg + pVPg and pRHDV-luc/ΔVPg were compared using SAS 9.1 software. The Student’s t test and ANOVA were used for the statistical analyses. P <0.05 was considered as significantly different (*), and P <0.01 was considered as extremely significant different (**).The experiments were conducted in triplicate, and similar results were obtained from three independent experiments.