Establishment of a neutralization assay for Nipah virus using a high-titer pseudovirus system.

ABSTRACT:

Objective

To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities.

Methods

Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72 h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus.

Results

A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 10⁵ median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively.

Conclusions

A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.

SUBMITTER: Luo X 

PROVIDER: S-EPMC9860241 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature