Study on the recovery of CTN-1V5 strain rabies virus with reverse genetics system

Abstract

Use molecular cloning techniques. The CTN-1V5 strains of rabies virus genome is divided into six fragments in order to cloned into Eukaryotic expression vector PVAX 1.0(+). Recombinant full length genomic cDNA was flanked by a hammerhead ribozyme and hepatitis delta virus ribozyme. four helper plasmids encoding the nucleoprotein, phosphoprotein,Glycoprotein and the large protein were constructed and co-transfected with a plasmid containing the full-length CTN viral genome into Vero cells. Recombinant CTN-1V5 virus was successfully recovered from the cloned cDNA under the control of a CMV promoter driven by RNA polymerase II. The recovered CTN-1V5 virus was identified by direct Immunofluorescence technique with anti-nucleoprotein monoclonal-antibody. A group of mice was challenged with the recombinant-strain by intracerebral inoculation, resulting in 100% morbidity.and the titer of the recombinant CTN-1V5 virus was 5.0 log LD50/mL.