Development of a reverse genetics system for a human rabies virus vaccine strain employed in China

The CTN rabies virus was isolated from a human in China in 1953 and subsequently attenuated by multiple passaging to a vaccine strain now approved by the WHO. In this study, we describe the development of a reverse genetics system for the CTN rabies virus strain. The recombinant full-length genomic cDNA was flanked by a hammerhead ribozyme (HamRz) and the hepatitis delta virus ribozyme (HdvRz) while the non-coding G-L region was replaced with a green fluorescent protein (GFP) gene. A set of helper plasmids encoding nucleoprotein (N), phosphoprotein (P), and large protein (L) were constructed and co-transfected with recombinant full-length genome plasmid into BHK-21 cells. Recombinant virus was successfully recovered from cloned cDNA under control of the CMV promoter driven by RNA polymerase II. The recombinant virus, CTN-GFP, stably expressed GFP as detected by fluorescence microscopy. A group of 1-day-old suckling mice was challenged with the CTN-GFP strain by intracerebral inoculation, resulting in 100% morbidity and GFP expression was detected in brain tissues. The recombinant virus CTN-GFP strain recovered from cloned cDNA will be useful as a viral vector to express other foreign genes.