A simplified method for measuring neutralising antibodies against rabies virus

Area of Science:

• Virology
• Immunology
• Biotechnology

Background:

• Rabies virus (RABV) causes fatal encephalitis, preventable by vaccination.
• The fluorescent antibody virus neutralization (FAVN) test measures rabies virus neutralizing antibodies.
• FAVN involves live virus incubation, cell fixation, and FITC-conjugated antibody staining for microscopy.

Purpose of the Study:

• To develop a simplified and more efficient method for measuring rabies virus neutralizing antibodies.
• To construct a recombinant fluorescent rabies virus for direct visualization of infected cells.
• To validate the use of this new recombinant virus as an alternative to the standard FAVN test.

Main Methods:

• Reverse genetics was used to create a recombinant RABV (mCCCG) expressing mCherry fluorescent protein.
• The glycoprotein of the recombinant virus was replaced with that of the Challenge Virus Standard (CVS)-11 strain for antigenic authenticity.
• In vitro growth kinetics and viral stability across passages were assessed.
• Comparative analysis of the neutralization test using mCherry producing virus (NTmCV) against the FAVN test was performed.

Main Results:

• The mCCCG virus expressed mCherry protein effectively, allowing direct observation of infected cells.
• In vitro growth kinetics of mCCCG were comparable to CVS-11.
• The recombinant virus demonstrated good stability through multiple passages.
• NTmCV showed equivalent results to the FAVN test for measuring rabies virus antibody titers.

Conclusions:

• The mCCCG virus is a suitable alternative to CVS-11 for rabies virus antibody titration.
• NTmCV simplifies the assay by eliminating the need for antibody conjugates and reducing assay time.
• This method is particularly beneficial for rabies serological assessment in resource-limited settings.
• Automated plate reading using a cell imaging reader is possible with NTmCV.