Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10 9.8 50% egg infective doses (EID 50 )/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10 8.3 EID 50 completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.

The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (G R333Q ) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the G R333Q mutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.Rabies virus (RV) belongs to the genus Lyssavirus of the family Rhabdoviridae and causes a fatal neurological disease in humans and animals (6). The RV genome is a nonsegmented negative-strand (NNS) RNA encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase (L). Among these, the G protein is a major contributor to RV pathogenicity (7,31,33). The G protein facilitates fast virus entry and transsynaptic spread and regulates the rate of virus replication, together with other viral elements (8,30,39). The G protein of nonpathogenic RV strains can trigger apoptosis, while the RV G of pathogenic strains induces less or no apoptosis (35, 59). The amino acid residue at position 333 of the G protein (G333) of some fixed strains has been shown to be an important determinant of virulence in adult mice (5). Strains that have arginine or lysine at position G333 kill adult mice, whereas mutants with other amino acids at this site cause a nonlethal infection (1,5,25,36,49,53). However, the pathogenicity of RV strains is not solely determined by substitutions at the G333 position. Other substitutions in the G protein, such as N194K, have also been shown to affect viral pathogenicity in mice (10, 21, 50). In addition, other viral elements, such as the N, P, M, and L genes, the trailer sequence in the noncoding region, and the pseudogene, were also reported to modulate RV pathogenicity (12,46,57,58). How these viral elements regulate the pathogenicity of RV remains to be fully explored, and further investigation is needed to understand the molecular basis of RV pathogenicity.Attenuated Flury RV low-egg-passage (LEP) and high-eggpassage (HEP) strains were established through serial passage in chicken brain, chicken embryos, and culture cells using a Flury RV isolated from a girl who died of rabies (23,24). LEP has Arg at position G333 and kills adu...

The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.