Nipah virus-derived defective interfering particles generated using non-infectious viral replicon particles confer protective efficacy comparable to those produced with standard full-length infectious virus.

Defective interfering particles (DIPs) are naturally occurring virus-like particles containing truncated fragments of the parental viral genome and have gained interest as potential medical countermeasures (MCMs) against viral infections. Using the Syrian hamster model of Nipah virus (NiV) disease, we previously showed that treatment with artificially produced NiV-derived DIPs markedly reduced clinical signs and mortality. However, DIP production required infectious NiV and BSL-4 containment, creating a major barrier to clinical translation. Here, we describe an improved, non-infectious, BSL-2-compatible NiV replicon system for DIP generation that eliminates the need for infectious virus. DIPs produced using this system inhibited virus in vitro and retained full protective efficacy in hamsters. By removing the requirement for high-containment virus, this approach overcomes key regulatory and practical hurdles, enabling advancement of DIP-based therapeutics toward clinical evaluation and eventual use against NiV and related henipaviruses.