Recombinant Canine Distemper Virus Snyder Hill Strain Expressing Glycoprotein Of Rabies Virus

Rabies is a fatal zoonosis characterized by acute and progressive meningoencephalitis and is caused by rabies virus(RV).It kills about 59,000 people and hundreds of thousands of animals annually.More than 95% of the human deaths occur in Asia and Africa,and about 99% of rabies human cases originate from rabid dogs.Dog rabies control measures substantially reduce human exposure and can be accomplished through dogs vaccination.Only if the immunization coverage reaches 70% the dog rabies can be controlled and eliminated potentially.For the reason that live attenuated rabies vaccines are prohibited from producing and using,and the inactivated ones are safe,effective,but costly.Therefore,it is essential to develop an effective,safe and cheap rabies vaccine.Canine distemper(CD)is caused by canine distemper virus(CDV)and characterized by seasonal epidemics.CD can be widespread in canines,mustelidae,raccoon and other carnivorous animals.The host range of CDV is constantly expanding worldwide and posing a serious threat on the development of animal husbandry.It is proved that attenuated vaccine can prevent the spread of CD in carnivorous animals effectively.And the development of reverse genetic technology makes it possible for CDV as a vaccine vector.In this study,we established a reverse genetics system of CDV Snyder Hill Strain isolated by our laboratory,and successfully rescued r CDV which maintained similar growth characteristics in Vero cells compared with wild type CDV Snyder Hill strain.And the r CDV's titer reached the peak of 1×106.1 TCID50/m L at 120 h post-infection.To investigate the potential of CDV serving as a live vaccine vector,we constructed a recombinant virus r CDV-EGFP by inserting EGFP gene between P and M genes of CDV genome.We found that the r CDV-EGFP had a similar biological characteristics with r CDV including excellent adaptability and genetic stability in Vero cells.After 18 generations' subculture,r CDV-EGFP still had ability to express EGFP effectively in Vero cells.Further,we constructed and rescued the r CDV-RVG which expressed the glycoprotein of RV correctly and stably.It was proved that the r CDV-RVG had a similar growth curve with r CDV and reached the peak titer of 1×105.7 TCID50/m L at 120 h post-infection.The results of immunity tests showed that the dogs inoculated with r CDV-RVG at the dose of 105.5 TCID50/m L produced specific neutralizing antibodies against RV and CDV respectively.The average level of RV neutralizing antibodies was 21.71 IU/m L,which was beyond the threshold of RV protective neutralization antibody 0.5 IU/m L.In conclusion,the results show that the recombinant canine distemper virus r CDV-RVG expressed RVG protein is a candidate vaccine against rabies.