Design optimization and characterization of a dual-reporter Nipah virus pseudovirus

Nipah virus (NiV), a highly pathogenic agent causing severe neurologic and respiratory disease, requires Biosafety Level 4 (BSL-4) containment. While existing pseudovirus models (e.g., based on human immunodeficiency virus (HIV) or vesicular stomatitis virus (VSV) backbones) offer alternatives, their utility is often limited by reliance on a single reporter gene. Here, we developed and optimized a robust NiV pseudovirus system utilizing a VSV-ΔG backbone co-expressing dual reporters, Green Fluorescent Protein (GFP) and NanoLuciferase (NLuc). Preliminary characterization showed that VSV-ΔG-GFP/NLuc exhibited structural integrity, achieved infection rates up to 70% in susceptible cells, and demonstrated relative stability within five freeze-thaw cycles. The study on the correlation between the expression of ephrin-B2 (EFNB2) in various cell lines and the infection efficiency of pseudovirus indicates the effectiveness of the pseudovirus. Furthermore, the platform reliably detected dose-dependent neutralization by established monoclonal antibodies targeting the fusion protein and glycoprotein of NiV, yielding trends comparable to authentic virus neutralization. Collectively, these findings establish this dual-reporter NiV pseudovirus as a safe, scalable, and high-throughput tool for antiviral screening and mechanistic studies under BSL-2 conditions. The synergistic combination of GFP (enabling visual quantification and sorting) and NLuc (providing ultra-sensitive, broad dynamic range quantification) enhances both versatility and data reliability. This study not only provides a critical new tool for NiV research but also serves as a valuable reference for developing pseudovirus platforms targeting other enveloped viruses.