Construction Of A Recombinant Newcastle Disease Virus Expressing Rabies Virus Glycoprotein And Research Of Immunogenicity

Rabies, also known as hydrophobia, is caused by the rabies virus(RV), which against central nervous system with acute viral human zoonotic diseases. Human rabies is by far the highest fatality rate of acute infectious diseases, once infected, the mortality rate of up to 100%. Rabies is mainly distributed in Asia, Africa and Latin America and other developing countries. In recent years, the incidence of rabies in China was constantly on the rise, in the world and second only to India. Epidemiological investigation showed that: 95% of human rabies cases are caused by direct contact of sick or infected dogs or cats.Glycoprotein of rabies virus (RVG) is the most in-depth protein, which researched in structure and immunology. RVG is a transmembrane protein to form trimer located on the surface of the virus as spikes and the major surface antigen of the virus. RVG is a ligand between the viral and host cell binding, to interaction with cell surface receptors, mediate the combination of virus and target cells. RVG, not only with the virulence of the virus, closely related to pathogenicity, but also the effective protection of rabies virus antigen, can induce neutralizing antibodies and stimulate cellular immunity, protecting the body against infection.Negative strand RNA virus reverse genetics system (RGS) is succeed to establish the Newcastle disease virus (NDV) as the most value of the live virus expression vector. After years of clinical application, it showing a remarkable advantage: genetic stability, NDV is very unlikely to recombine in strains and return strong of virulence; NDV occurred only a transient infection in mammals and humans, without potential risk for human; NDV has a high titer growth characteristics in chicken embryo and low cost, easy to scale production. As a vaccine vector, NDV has a broad application space and great economic significance.Rescue of a recombinant Newcastle disease virus expressing rabies virus glycoprotein with NDV reverse genetic system (RGS) and research of immunogenicity in mice. The RVG gene was generated from ERA vaccine strain of rabies virus by PCR with specific primers and cloned into pBRN-FL-PmeI,constructing a recombinant NDV which expressed the glycoprotein of rabies. The recombinant virus was rescued by calcium phosphate transfection method with helper plasmids including pBS-NP, pBS-P and pBS-L. BALB/c mice vaccinated with rL-RVG at dose of 2×108EID50 by intramuscular injection and boosted after 21d. RVG gene was detected by RT-PCR in allantoic fluid of 10-day-old embryonated SPF chicken eggs. The results of western blot and indirect immunofluorescence assay (IFA) confirmed that RVG gene had been expressed. BALB/c mice vaccinated with rL-RVG didn't show any disease signs and weight lose and fully protected from challenge with 50 MLD50 of GX/09, 14 days after second immunization. Continuous monitoring of specific neutralizing antibody level of ERA in Beagle, the results show that with good immunogenicity. This work provided the basis for the development of a novel live viral vector vaccine to prevent rabies.