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  • Title: Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay.
    Author: Yang DK, Kim HH, Park YR, Yoo JY, Park Y, Park J, Hyun BH.
    Journal: J Vet Sci; 2021 Jul; 22(4):e56. PubMed ID: 34313041.
    Abstract:
    BACKGROUND: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. OBJECTIVES: We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). METHODS: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. RESULTS: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (108.0 TCID50/mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). CONCLUSIONS: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.
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