Construction And Immunogenicity Of Rabies Virus Glycoprotein Recombinant Pseudorabies Virus Mediated By Transposon

Rabies, a neurotropic infectious disease caused by rabies virus belonging to genus Lyssavirus within the family Rhabdoviridae, still breaks out occasionally in more than80countries around the world even today. This disease was firstly known over3000years ago and leads to50000to55000deaths annually especially in developing countries, among which3000cases occur in China, with95%transmitted by virus-carrying canines.The best way to control rabies is to vaccinate dogs with effective vaccines. However, although commercial vaccines with high safety and efficiency play a significant role in preventing and controlling rabies, yet, in developing countries, they were not widely applied because the high expenses and the repeat immunization for getting effective protection. Besides, for the various problems of the late-model vaccines they could not be practically used till now. As a result, developing a kind of high-efficiency and low-cost rabies vaccine becomes urgent.Pseudorabies virus (PRV) was considered as a high-perspective vaccine vector because of its good genetic stability, large exogenous gene capacity, high progeny virus titer and broad host range, including sheep, cattle, dogs, and rodents, but not human.In our research, a recombinant herpesvirus type I harboring the glycoprotein gene of rabies virus (RABV) was constructed by means of transposon-mediated gene transfer. We used PRV Bartha-K61vaccine strain as the vector, and constructed a recombinant pseudorabies virus (rPRV/SRV9G/EGFP) by Tn5transposon expressing the glycoprotein of rabies virus SRV9strain and EGFP, first time and carried out the immunity test in cats.First, a double expressing cassette of SRV9G and EGFP was constructed, and then, was cloned into the Tn5transposon vector pUCMOD. Second, The transposon fragment including the double expressing cassette was inserted in vitro into genome DNA of PRV Bartha catalyzed by Tn5transposase. Third, transposed PRV genome DNA was transfected into BHK cells to form a pool of recombinant PRV. Forth, recombinant PRV was purified using picking the plaque cells that shining green light under the fluorescence microscope. Finally, after6cycles of purification, we got the recombinant PRV--rPRV/SRV9G/EGFP.The detail progress of getting the recombinant pseudorabies virus could be summarized as follows:at first, we constructed a double expressing cassette of SRV9G and EGFP and cloned it into the Tn5transposon vector pUCMOD; then the transposon fragment including the double expressing cassette was inserted in vitro into the genome DNA of PRV Bartha catalyzed by Tn5transposase, the transposed PRV genome DNA was transfected into BHK cells to form a pool of recombinant PRV. After6cycles of plaque purification we got the recombinant PRV--rPRV/SRV9G/EGFP.The recombinant virus was identified to have the gene incorporated into the gI gene of the herpesvirus genome. The result of transcription, expression of foreign genes and genetic stability of recombinant virus test showed that the foreign genes could be correctly expressed and react with the RABV positive serum. The recombinant virus were genetic stability when they were passaged to30th generations on BHK cells.Identification test showed the target gene have been incorporated into the gI gene of the herpesvirus genome, and could be expressed stably in the recombinant pseudorabies virus. At the same time, the recombinant virus could be recognized by RABV positive serum. Genetic stability was also identified after cultured on BHK cells for30generations.The cats were inoculated with the recombinant virus by intramuscular injection or oral vaccination. The results indicated the recombinant virus could induce antibody targeting the glycoprotein of the rabies virus, which could be tested through Western blot and Mouse intracerebral. For the neutralization test, the neutralizing antibody titers of all cats with intramuscular inoculation and oral vaccination after4weeks post-vaccination were reached>0.5IU/mL, which is the universally accepted protective level. The result also indicated that the pseudorabies virus vector could infect dogs and replicate in vivo, and that the rabies virus glycoprotein had been expressed and elicited an effective immune response. The high antibody titers were maintained for over3months. No side effects were observed in both the orally and intramuscularly inoculated cats. These results indicated that the recombinant herpesvirus expressing the glycoprotein of RAB V may serve as an efficient oral vaccine for cats, especially for the large amount of stray cats in cosmopolitan areas.