Molecular Basis of Neurovirulence of Flury Rabies Virus Vaccine Strains: Importance of the Polymerase and the Glycoprotein R333Q Mutation

Abstract

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...

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