Generation of Recombinant Rabies Virus CVS-11 Expressing eGFP Applied to the Rapid Virus Neutralization Test

Xianghong Xue,Yuwei Gao,Xuexing Zheng,Yongkun Zhao,Xianzhu Xia,Hongru Liang,Na Feng,Songtao Yang,Hualei Wang

doi:10.3390/v6041578

Abstract

The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

Highlights

Rabies is a fatal zoonotic infectious disease caused by the rabies virus (RABV) that affects numerous warm-blooded mammals worldwide [1]
To verify the rescued RABV rCVS-11-enhanced green fluorescent protein (eGFP) strain replication and expression of eGFP as an RABV structural protein, Baby hamster kidney (BHK)-21 cells infected with rCVS-11-eGFP were immunostained with
To confirm that the rCVS-11-eGFP strain was derived from the full-length plasmid pCVS-11-eGFP, BHK-21 cells infected with the rCVS-11-eGFP strain were used to perform

Summary

Introduction

Rabies is a fatal zoonotic infectious disease caused by the rabies virus (RABV) that affects numerous warm-blooded mammals worldwide [1]. Dogs and cats are the major reservoir of human rabies in most parts of the developing world [5], and the vast majority of human rabies victims become infected by dog bites [6,7]. 70% immunization of the dog population can efficiently block RABV transmission [4,8], and that virus-neutralizing antibody plays an important role in protecting against RABV [9,10]. Quantification of the VNA levels against RABV is essential for the timely monitoring of immunization coverage in dog and cat populations and to evaluate the immunity effectiveness of RABV vaccines

Results

Discussion

Conclusion