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

Abstract

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.

Keywords: Arctic-related clade; Komatsugawa; fixed virus; pathogenesis; rabies virus; street virus.

Figure A1

Distribution of viral antigens in the amygdala, cerebral neocortex, and hippocampus in mice infected with the virus strains. The distribution of viral antigens in the brain was re-examined by using each of two brain samples infected with the Koma, 1088, or RABV-Dog strain. Viral protein and nuclei were stained with an anti-RABV P protein antibody (green) and DAPI (blue), respectively. The scale bars correspond to 100 µm. These samples were photographed by using the Biozero fluorescence microscope BZ-8000 series (Keyence).

Figure 1

Schematic diagram of the genome organization of the Koma strain. Boxes and lines show open reading frames and non-coding regions, respectively. nt: nucleotides, aa: amino acids.

Figure 2

Phylogenetic relationships of the Koma strain with other rabies virus (RABV) strains. Complete genome nucleotide sequences of a total of 63 strains, which are representative strains of the major phylogenetic clades previously reported [31], were obtained from the GenBank database and were subject to phylogenetic analysis by the maximum-likelihood method, together with the genome sequence of the Koma strain. The genetic analysis software Molecular Evolutionary Genetics Analysis (MEGA) version 10.1 was used for phylogenetic analysis. Numbers at nodes and in parentheses represent bootstrap values obtained from 1000 replicates and the accession numbers in the GenBank database. The bar indicates 0.10 substitutions per nucleotide position. The Koma strain and two street strains (1088 and RABV-Dog) are shown in red and blue, respectively.

Figure 3

Amino acid substitutions in N, P, M, G, and L proteins between Koma and RV303. Vertical bars with numbers on each protein indicate positions of the substitutions. Amino acid residues of the Koma strain (upper) and RV303 strain (lower) at the substitution sites are shown as a single letter code. The amino acid number in the G protein is assigned to the mature form that does not contain an N-terminal signal peptide of 19 aa (shaded).

Figure 4

Positions and total numbers of potential N-glycosylation sites in the G protein of RABV strains. Black triangles with numbers represent the potential N-glycosylation sites. Information on the glycosylation sites in the street and fixed strain G proteins was obtained from articles by Yamada et al. [23] and Hamamoto et al. [47].

Figure 5

Growth curves for the Koma strain and representative street and fixed strains in mouse neuroblastoma C1300 (NA) cells. NA cells were inoculated with each of the virus strains at an MOI of 0.001. Viruses in the culture supernatants were collected at 0, 1, 3 and 5 dpi and titrated in NA cells by a focus assay. This assay was performed in triplicate, and the values in the graph are shown as means ± standard errors of the means. *, Significant differences vs. the Koma strain at a p value of < 0.05; ns, not significant (p ≥ 0.05).

Figure 6

Focus formation by Koma and representative street and fixed strains in mouse neuroblastoma C1300 (NA) cells. (A) At 3 days after inoculation of NA cells with each virus strain at an MOI of 0.0002, the cells were fixed and immunostained with an anti-RABV N protein antibody. The scale bars correspond to 200 µm. (B) Fifty foci of each strain were randomly selected to quantify their areas by Image J software. Each column represents the average area (± standard errors of the means). *, Significant differences vs. the Koma strain at a p value of < 0.05; ns, not significant (p ≥ 0.05).

Figure 7

Incubation periods in mice infected with virus strains via the i.m. route. Six-week-old male mice (10 mice/group) were inoculated intramuscularly with 3.0 LD₅₀ of each of the strains into the left thigh muscle and were observed daily for 50 days. Each column represents the date of onset of disease in each mouse (mean ± standard deviation). *, number of sick/inoculated mice.

Figure 8

Distribution of viral antigens in the brains of mice infected with the virus strains. The brains were collected from mice infected with each virus strain at the terminal stage of infection. A mock-infected brain was collected at 35 dpi. The brain tissues were stained with an anti-RABV P protein antibody. (A) Representative immunofluorescence staining image of RABV P protein (green) in the whole brains of infected mice. The scale bars correspond to 3.5 mm. (B) Magnified fields of the amygdala, cerebral neocortex, and hippocampus in mice infected with Koma, 1088, RABV-Dog, or Mock. Nuclei were stained with DAPI (blue). The scale bars correspond to 150 µm.