Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein.

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.

Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s.

The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.