Clin Exp Vaccine Res.  2016 Jul;5(2):159-168. 10.7774/cevr.2016.5.2.159.

Safety and immunogenicity of recombinant rabies virus (ERAGS) in mice and raccoon dogs

  • 1Viral Disease Division, Animal and Plant Quarantine Agency, MAFRA, Gimcheon, Korea.
  • 2College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
  • 3Jeonnam Wildlife Management Center, Suncheon, Korea.


The development of a genetically modified live rabies vaccine applicable to wild raccoon dogs is necessary for the eradication of rabies in Korea. Thus, we constructed a recombinant rabies virus (RABV) called the ERAGS strain, using a reverse genetic system and evaluated its safety and efficacy in mice and its safety and immunogenicity in raccoon dogs.
ERAGS, which has Asn194Ser and Arg333Glu substitutions in the glycoprotein, was constructed using site-directed mutagenesis. Mice were inoculated with the ERAGS strain (either 10(5.0) or 10(7.0) FAID(50)/mL) via intramuscular (IM) or intracranial injections and then challenged with a virulent RABV. Raccoon dogs were administered the ERAGS strain (10(8.0) FAID(50)/mL) either orally or via the IM route and the immunogenicity of the strain was evaluated using fluorescent antibody virus neutralization tests.
The ERAGS strain inoculated into murine neuroblastoma cells reached 10(7.8) FAID(50)/mL at 96-hour post-inoculation. The virus was not pathogenic and induced complete protection from virulent RABV in immunized 4- and 6-week-old mice. Korean raccoon dogs immunized with the ERAGS strain via IM or oral route were also safe from the virus and developed high titer levels (26.4-32.8 IU/mL) of virus-neutralizing antibody (VNA) at 4 weeks post-inoculation.
The ERAGS RABV strain was effectively protective against rabies in mice and produced a high VNA titer in raccoon dogs.


Rabies virus; Mouth; Vaccines; Raccoon dogs

MeSH Terms

Mutagenesis, Site-Directed
Neutralization Tests
Rabies Vaccines
Rabies virus*
Raccoon Dogs*
Rabies Vaccines


  • Fig. 1 Full-length plasmid map for the construction of the ERAGS strain. The full-length cDNA plasmid was modified using site-directed mutagenesis at positions 194 and 333 of the glycoprotein in the ERA strain.

  • Fig. 2 Cytopathic effects (×200) of BHKT7-9 cells (A) infected with the ERAGS strain and normal cells (B). BHKT7-9 cells infected with the virus were detached from the plate.

  • Fig. 3 Immunofluorescence (×200) of the ERAGS strain using an indirect fluorescent assay test with monoclonal antibodies against the nucleoprotein of the rabies virus (A) and normal cells (B). Recombinant rabies virus-specific fluorescent sites appeared in the cytoplasm of the infected BHK/T7-9 cells.

  • Fig. 4 Results of the rapid immunodiagnostic assay (RIDA) kit following the application of the ERAGS strain supernatant. The "C"and "T"in the RIDA kit stand refer to the "control"and "test"lines. The appearance of two lines was considered to be a positive result.

  • Fig. 5 Multistep growth curves of the ERA and ERAGS strains in NG108-15 cells. Both strains revealed the highest viral titer at 96-hour post-inoculation.

  • Fig. 6 Changes in body weights in 4-week-old (A) and 6-week-old (B) mice inoculated with either the ERA or ERAGS strains via either the intramuscular (IM) or intracranial (IC) route. The weights of mice inoculated with the ERAGS strain continued to increase whereas those of mice inoculated with ERA progressively decreased, and these mice eventually died.

  • Fig. 7 Survival in 4-week-old (A) and 6-week-old (B) mice inoculated with either the ERA or ERAGS strain via either the intramuscular (IM) or intracranial (IC) route. Mice inoculated with ERA (both IM and IC) died 10 and 16 days post-inoculation, respectively. In contrast, 100% of mice inoculated with ERAGS (both IM and IC) survived, as did controls.

  • Fig. 8 Changes in body weights (A, B) and survival rates (C, D) in 4- and 6-week-old mice that were immunized with the ERAGS strains via either the intramuscular (IM) or intracranial (IC) route and then challenged with a highly pathogenic rabies virus strain (CVSN2c). The average body weight increased for 14 days after the challenge and the survival rate did not change for 14 days after the challenge.

  • Fig. 9 The immune responses and body weights of raccoon dogs that received the ERAGS strain via either the oral or intramuscular (IM) route (26.4 and 32.8 IU/mL, respectively). At 4 weeks post-inoculation, high virus-neutralizing antibody titers were induced in all immunized raccoon dogs (A). The body weights of those that received oral administrations significantly increased whereas those that received IM administrations did not (B). Each bar represents the mean±standard error of the mean of three independent samples. Different lowercase letters indicate significant differences (p < 0.05, Tukey's post hoc test). Asterisks (*p < 0.05) and double asterisks (**p < 0.01) in each panel indicate significant differences from corresponding control groups without any treatment (unpaired t-test). FAVN, fluorescent antibody virus neutralization.

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Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein
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J Vet Sci. 2020;21(2):e22.    doi: 10.4142/jvs.2020.21.e22.

A genetically modified rabies vaccine (ERAGS) induces protective immunity in dogs and cattle
Dong-Kun Yang, Ha-Hyun Kim, Seung Heon Lee, Woong-Ho Jeong, Dongseop Tark, In-Soo Cho
Clin Exp Vaccine Res. 2017;6(2):128-134.    doi: 10.7774/cevr.2017.6.2.128.

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein
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J Vet Sci. 2020;21(2):.    doi: 10.4142/jvs.2020.21.e22.


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