Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay
- Author:
Dong-Kun YANG
1
;
Ha-Hyun KIM
;
Yu-Ri PARK
;
Jae Young YOO
;
Yeseul PARK
;
Jungwon PARK
;
Bang-Hun HYUN
Author Information
- Publication Type:Original Article
- From:Journal of Veterinary Science 2021;22(4):e56-
- CountryRepublic of Korea
- Language:English
-
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 (108.0TCID50/mL) in VERO cells at 5 days postinoculation, 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.
