Fig. 1: Rescue of LGTV infectious clones by reverse genetics.
A Schematic illustration of RNA-based rescue of LGTV by amplification of two overlapping DNA fragments (fragment 1 and 2) to produce final DNA fragment 3 that comprises SP6 promoter and LGTV genome. The final DNA fragment was in vitro transcribed to produce LGTV RNA with a m7G(5´)ppp(5´)G cap before transfecting into cells. B Schematic illustration of DNA-based rescue of LGTV by four overlapping DNA fragments comprising cytomegalovirus promoter, LGTV genome, the hepatitis delta ribozyme and the simian virus 40 polyadenylation signal. The overlapping fragments were amplified by PCR and mixed in equimolar ratio before transfecting into cells. The viruses rescued by RNA- and DNA-based methods were passaged five times in cell culture. C Immunoblotting of cell culture media collected at day 7 from the transfected cells. Detection was performed using anti-LGTV E antibody. D Immunofluorescence assay of Vero E6 cells infected with LGTVRNA+L–2000. Staining was performed using anti-TBEV E primary antibody and Alexa 488 secondary antibody.
