Fig. 6.

Rescue efficiency of RNA/DNA-launched infectious system. (a) 4.0 μg of the DNA-launched infectious clones or in vitro transcribed product of the same amount of RNA-launched infectious clones were used for BHK-21 transfection. The DNA-launched infectious clone (plasmid pIR-DHAV-1) and the empty vector (plasmid pIR) were transfected directly into BHK-21 cells, while the RNA-launched infectious clone was transcribed in vitro, and then the transcription mixture was transfected into BHK-21 cells. At 48 hpt, IFA observation was conducted after staining with anti-DHAV-1 mAb 4F8 (dilution of 1:1000 with PBS) for 1 h at 37 °C, followed by incubation in 37 °C for 1 h with FITC-labeled goat anti-mouse antibody. Bars represent 10 μm. (b) Western blotting assay. A total of 4.0 μg of recombinant plasmid of DNA-launched infectious clone and in vitro transcribed product from 4.0 μg of RNA-launched infectious clone were transfected into BHK-21 cells to analyze rescue efficiency between DNA-launched system and RNA-launched system. Cell culture was collected at 48 hpt and run on SDS 12%-polyacrylamide gels. Anti-DHAV-1 monoclonal antibody 4F8 (dilution of 1:500) and HRP-conjugated goat anti-mouse antibody (dilution of 1:3000) were used to conduct the western blot assay. BHK-21 cells that transfected with pIR vector were conducted as negative control. (c) BHK-21 cells were infected with transfected product of 4.0 μg of the DNA-launched infectious clones or in vitro transcribed product of the same amount of RNA-launched infectious clones, cell lysates were collected at 48 hpi, and were immediately used for RNA extraction and qRT-PCR measurement. Viral RNA copies were calculated with formula X = 6.7 × 10(40.812-y)/3.285. “X” is a standard of viral copies while “y” is a standard of values derived from one step real-time PCR. Bars represent means and standard of three individual repeats. “***” represents p < 0.001