Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system

Jianhui Nie,Weijin Huang,Shouchun Cao,Qiang Liu,Jian Ma,Xiaohong Wu,Yuhua Li,Xuguang Li,Youchun Wang

doi:10.1038/srep42769

Abstract

Pseudoviruses are useful virological tools because of their safety and versatility; however the low titer of these viruses substantially limits their wider applications. We developed a highly efficient pseudovirus production system capable of yielding 100 times more rabies pseudovirus than the traditional method. Employing the high-titer pseudoviruses, we have developed robust in vitro and in vivo neutralization assays for the evaluation of rabies vaccine, which traditionally relies on live-virus based assays. Compared with current rapid fluorescent focus inhibition test (RFFIT), our in vitro pseudovirus-based neutralization assay (PBNA) is much less labor-intensive while demonstrating better reproducibility. Moreover, the in vivo PBNA assay was also found to be superior to the live virus based assay. Following intravenous administration, the pseudovirus effectively infected the mice, with dynamic viral distributions being sequentially observed in spleen, liver and brain. Furthermore, data from in vivo PBNA showed great agreement with those generated from the live virus model but with the experimental time significantly reduced from 2 weeks to 3 days. Taken together, the effective pseudovirus production system facilitated the development of novel PBNA assays which could replace live virus-based traditional assays due to its safety, rapidity, reproducibility and high throughput capacity.

Highlights

Pseudoviruses are useful virological tools because of their safety and versatility; the low titer of these viruses substantially limits their wider applications
While it is likely that chimeric glycoproteins with VSV-G could partially resolve transduction issue[27], the use of chimeric glycoprotein would compromise the application of such pseudovirus to the analyses of neutralizing antibody targeting the envelope of the wild type virus
Three additional fluc expressing plasmids containing different promoters were constructed by replacing the CMV promoter of the pcDNA3.1.fluc with complete CMV promoter, CAG or LTR to generate psCMV.fluc, pCAG.fluc or pLTR.fluc, respectively. 293T cells were transfected with the aforementioned four plasmids, with relative light units (RLU) being analyzed 48 hours post-transfection

Summary

Introduction

Pseudoviruses are useful virological tools because of their safety and versatility; the low titer of these viruses substantially limits their wider applications. Pseudotyping of lentiviral vectors with RABV glycoprotein G has been explored in experimental gene therapy against neurological disorders[20,21,22] and determination of neutralizing antibody[23,24]; low transduction efficiency associated with the glycoprotein G pseudotyped virus substantially hinders its wider applications[25,26]. This is especially true for in vivo animal studies, in which no rabies pseudovirus has been reported. Novel approaches should be explored to circumvent these technical difficulties

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Results

Conclusion