Study On The Construction And Application Of Recombinant Rabies Virus Expressing West Nile Virus Envelope Protein

The West Nile virus disease is an acute zoonoses caused by West Nile virus(WNV). Nearly 20 years,WNV has spread worldwide,and has caused the outbreak of human and animal diseases, which has become a worldwide public health problem.In 2011, Liang Guodong from mosquitoes collected in China’s Xinjiang Uygur Autonomous Region collection separation to WNV, a large number of serological results show that local not only the existence of WNV disease caused by infection, also occurred in WNV infection caused by virus encephalitis in.So far, there have been no approved effective drugs or specific vaccines of WNV disease.In this study, using the reverse genetics system of rabies virus(RABV) SRV9 strain, the E gene of WNV was inserted between P-M genes of SRV9 by PCR to constructthe full-length plasmid pD-SRV9-WNV-E. To rescue the recombinant RABV(rSRV9-WNVE), BSR cells were transfected with the full-length plasmid together with helper plamids(nucleoprotein, phosphoprotein, large transcription protein and glycoprotein)using Lipofectamine 3000 Transfection Reagent.A direct immunofluorescence assay showed that rSRV9-WNVE could be rescued successfully. The indirect immunofluorescence, western blot and PCR results showed that rSRV9-WNVE could successfully express WNV-E protein.In addition, the growth curve showed that the growth characteristics of the recombinant virus could not be influenced by the exogenous gene expression.The mice were infected with parent virus SRV9 and rSRV9-WNVE throught an intracerebral route.The mice were observed daily for development of rabies and survivorshipfor 3 weeks. Safety test showed that rSRV9-WNVE was highly safe to mice for which pathogenicity was significantly decreased.The mice were vaccinated with 5×105 and 5×106 of each virus(SRV9 and rSRV9-WNVE) through an intramuscular route.and booster immunized with the same dose and route. Mice serum were collected at two weeks post vaccinated and the virus neutralizing antibodies(VNA) against RABV and antibodies against WNV were detected.The results showed that rSRV9-WNVE could induce both VNA against RABV and specific antibody against WNV. It indicated that rSRV9-WNVE had a good immunogenicity.In conclusion, rSRV9-WNVE was safe and had a good safety and immunogenicity to mice, with the potential to be used as a vaccine,with lays the foundation to develop a new vaccine of WNV.