Fig. 4

Identification of the rescued virus. (a) Western blotting assay. The BHK-21 cells infected with parental virus and rescued viruses were harvested at 48 hpi. 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. The BHK-21 cells transfected with pIR vector were used as negative control. (b) Identification of the genetic marker in the rescued virus. The BamH I restriction enzyme site was introduced into the recombinant plasmid to create a genetic marker to distinguish the rescued virus from the parental virus (without BamH I enzyme site). Two 2290 bp-fragments derived from parental and rescued virus with primers DHAV-3F and DHAV-4R were digested with BamH I and analyzed on a 2.0% agarose gel. (M) DNA Marker DL2000; 1. Fragment amplified with template of parental RNA; 2. Fragment derived from the parental virus digested with BamH I; 3. Fragment derived from the rescued virus digested with BamH I; 4. Fragment amplified with template of rescued virus RNA