Reproducible generation of Nipah virus pseudovirions with uniform incorporation of F and G surface glycoproteins for high-throughput neutralization assays

Nipah virus (NiV) is a pathogen to be handled in BSL-4 facilities. Multiple surrogate systems such as virus-like particles (VLPs) and pseudoviruses enable carrying out NiV neutralization assays and study of virus entry pathways in biosafety level-2 (BSL-2) facilities. These are dual protein expression and surface display systems comprising NiV structural glycoproteins F and G as the key components. During generation of NiV VLPs or pseudovirions, ensuring batch to batch uniformity is a major concern due to the lack of proportionate incorporation of these proteins in the producer cells as well as their consistent incorporation in the particles. We established HEK293 pseudovirion producer cells that stably co-incorporated NiV F and G proteins to address the issue. Fluorescence-activated cell sorting (FACS) analysis of clonally selected cells for high and uniform level F and G protein co-incorporation; and their further expansion were carried out to further refine the system. High titer vesicular stomatitis virus (VSV)-based pseudoviruses which showed consistent incorporation of both the glycoprotein were reproducibly generated from these producer cells. In functional assays, these Nipah pseudovirions (NiV) exhibited a dose-dependent neutralization by commercial anti-NiV F and G antibodies as well as by convalescent serum from Nipah recovered patients. A pseudovirus neutralization test (PVNT) with a secreted alkaline phosphatase (SEAP) as the reporter was established in the study. The assay supports high-throughput adaptability with a quick turn-around time. It will aid large-scale human and animal serosurveillance studies in Nipah endemic regions as well as screening of virus entry inhibitors and monoclonal antibodies.