J Vet Sci.  2021 Jul;22(4):e56. 10.4142/jvs.2021.22.e56.

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay

• Yang DK
• Kim HH
• Park YR
• Yoo JY
• Park Y
• Park J
• Hyun BH

• KMID: 2518802
• DOI: http://doi.org/10.4142/jvs.2021.22.e56

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 (10^8.0TCID₅₀/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.

Keyword

Rabies; green fluorescent protein; virus neutralizing antibody; FAVN