The Immunogenicity Of Recombinant New Castle Disease Virus Expressing Rabies Virus G Protein In Cattle And Sheep

Rabies caused by Rabies Virus(RV)infection is one of the most highly pathogenic zoonosis. The mortality rate is almost 100% once the clinical symptoms appeared. Development of a safe and effective vaccine is still required for reducing rabies outbreaks. RV is a single negative non-segment RNA virus from the genus Lyssavirus of the Rhabdoviridae family. RV genome mainly composed of five structural proteins:N、P、M、G、L. RV G protein is the main protein that can induce virus neutralizing antibody(VNA).Newcastle disease(NDV) is also non-segment negative single RNA virus. With the development of reverse genetics system, NDV strains have been developed as effective live-virus vectors. NDV has many advantages as live-virus vector : Firstly, NDV are effective in inducing serum, cellular, and mucosal immune responses when inoculated even in respiratory tract. Secondly, NDV usually do not integrate into the host genome so it is safe to the hosts. Finally, NDV can growth in very high titers in chicken embryos. This simplifies the vaccine production and reduced the costs of vaccine. We have generated a recombinant Newcastle disease virus(rLaSota)-vectored experimental rabies vaccine by expressing the G protein of RV strain ERA in previous study,We compared rLaSota and recombinant NDV rL-RVG for their spread in mammal cells. rLaSota can not spread from infect individual cells to adjacent BHK-21 cells without TPCK trypsin(1 mg/ml) as a low-pathogenicity NDV strain. IFA shown that rL-RVG can spread from cell to contiguous cell after 24 h postinfection in a similar manner with RV. rL-RVG shown the different diffusion mode from rLaSota but it did not increase the pathogenicity to the animals such as dogs, cats and mice. We further demonstrated that rL-RVG could induce high level rabies virus neutralizing antibody(VNA)in immunized animals(dog, cat and mice).We collected brain samples from suspected rabid animals in Inner Mongolia region, during 2011-2013. The virus were confirmed by DFA and RT-PCR and molecular epidemiologic analysis of all specimens demonstrated that rabies viruses in Inner Mongolia region have two different collum: local strains similar to those reported from previous epidemics in Hebei province, Shanxi province and Beijing and extraneous strains similar to those outbreak in Mongolia and Russia. the number of cattle and sheep rabies cases in Inner Mongolia region have increased during severe epidemics. We concern the safety and efficacy of rL-RVG as a recombinant vaccine to apply in cattle and sheep. In this study, we compared the sensitivity of rLaSota and rL-RVG to interferon and the ability to induce interferon on MDBK cells. Antiviral experimental results showed that rL-RVG is highly sensitive to interferon similar to its parental strain rLaSota. While the same does of rL-RVG induced sigifiantly higher level of interferon than that of rLaSota does. To determine the immunogenicity of G protein produced by rL-RVG, groups of cattle and sheep were immunized with rL-RVG via the intramuscular route. We confirmed the absence of RV-specific antibodies in serum samples collected from all preimmunized cattle and sheep. Cattle and sheep immunized with 109 EID50 and 108 EID50 of rL-RV did not show any clinical symptoms. Test of RV neutralizing antibody in blood samples from immunized cattle and sheep showed that rL-RVG can induce high level of VNA, and the titers of VNA raised rapidly after the boost immunization. The cattle inoculated with 108 EID50 induced higher titer of RV VNA than the group immunized with 109 EID50 after the second strengthen immune. After second immunization, protective neutralizing antibody could last for a long time in the cattle and sheep, and the antibody concentration stay higher than 0.5 IU(strong poison attack protective threshold)for 12 months after immunization. Our findings suggest that rL-RVG is an efficient and cost-effective vaccine produced in embryonated chicken eggs and has the potential to be used as a virus-vectored RV vaccine in cattle and sheep.