Fig. 1

Strategy for the construction of a full-length DNA-launched infectious clone based on a virulent strain LY0801 of DHAV-1. The fragment IR was derived from the plasmid pIRES2-EGFP (Clontech) using primers pIR-BamHI-XhoIF and pIR-AscI-BamHIR, and then was digested with BamH I and ligated with T4 DNA ligase to yield pIR vector. The LY0801 virus genome with two ribozyme sequences introduced at the both ends was placed downstream of the CMV promoter in pIR vector to construct the DNA-launched infectious clone, pIR-DHAV-1. A total of 4 overlapping fragments amplified from total RNAs extracted from the 5th passage of LY0801 virus were assembled successively into the pIR vector by unique restriction enzyme indicated for each fragment. A copy of hammerhead ribozyme sequence and Asc I restrict enzyme site were engineered at the 5′ end while a copy of hepatitis delta virus ribozyme (HDVRs) sequence and Xho I restrict enzyme site were engineered at the 3′ end of the cDNA copy of DHAV full-length genome, respectively. The base T at position of 3042 was mutated into base C to generate a BamH I restrict enzyme site which was treated as a genetic marker to distinguish the rescued virus from the parental virus