Establishment of a reverse genetics system for rabies virus strain Komatsugawa

Abstract

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.

Keywords: Japan; Komatsugawa; rabies virus; reverse genetics; street strain.

Fig. 1.

Detection of infectious rKoma rescued from cDNA by inoculation to mouse neuroblastoma (NA) cells. To rescue the rKoma from cDNA, BHK/T7-9 cells were transfected with the full-length genome plasmid together with four helper plasmids expressing N, P, G, and L proteins, according to the protocol for the rescue of the street 1088 strain [7]. At 4 days after transfection, the supernatant was collected and inoculated into NA cells. The NA cells at 6 days post-inoculation were fixed and immunostained with an anti-RABV N protein mouse monoclonal antibody 13–27 [17]. A negative control was prepared by inoculating with the supernatant from the recovery experiments without transfection with the L protein-expressing helper plasmid. The scale bars correspond to 200 μm.

Fig. 2.

Detection of genetic markers in the rKoma genome. (a) Schematic diagram of the cDNA fragment amplified by RT-PCR for checking the presence of the genetic markers (G-Mk#1 and 2) introduced into the rKoma genome. The sizes of the rKoma and wtKoma cDNA fragments after SpeI digestion are also indicated. (b) A cDNA fragment of 3,566 bp including the genetic marker sites was amplified by RT-PCR by using an RNA extract from an rKoma stock as a template. An RNA extract from a wtKoma stock was used for a positive control. M: molecular weight marker, +: with the RT step, -: without the RT step (c). The cDNA fragments from rKoma and wtKoma were subjected to sequencing by the conventional Sanger method after agarose gel purification. The genetic marker (G-Mk#1) in the rKoma gene is indicated on the nucleotide sequences. Nucleotide numbers are based on the genome nucleotide sequence of wtKoma [GenBank accession No. LC553558.1]. (d) The amplified cDNA fragments from rKoma and wtKoma were digested with SpeI before electrophoresis. M: molecular weight marker, +: cDNA fragments treated with SpeI, -: without SpeI.

Fig. 3.

Growth curves and focus sizes of rKoma and wtKoma in mouse neuroblastoma (NA) cells. (a) Growth curves of rKoma and wtKoma in neuroblastoma (NA) cells. Each virus was inoculated at a multiplicity of infection (MOI) of 0.001 into NA cells. Viral titers in culture supernatants collected at 1, 3, and 5 dpi were determined by a focus assay as reported previously [20]. This assay was carried out in triplicate. To check statistical significance, two-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test was conducted by using GraphPad Prism ver. 8.3 (GraphPad Software, San Diego, CA, USA). Error bars: means ± SEM, ns: not significant (P≥0.05). (b) Focus formation by rKoma and wtKoma in NA cells. NA cells were inoculated with each virus at an MOI of 0.0002 and then overlaid with eagle’s minimal essential medium (E-MEM) containing 1% methylcellulose. At 3 days after inoculation, the cells were fixed and immunostained with an anti-RABV N protein antibody 13–27. The scale bars correspond to 200 μm. (c) Quantification of the focus area. A total of 50 foci of each strain were randomly selected and photographed to quantify their areas by Image J software. Each column represents the average area. Student’s t-test was conducted by using GraphPad Prism to check statistical significance. Error bars: means ± SEM, ns: not significant (P≥0.05).