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.