De novo rescue of new henipaviruses under BSL-4 conditions – From sequence to pathogen

Zoonotic paramyxoviruses, including the highly pathogenic henipaviruses (HNVs), pose significant risks to global health due to their high mortality rates, potential for human-to-human transmission, and lack of approved countermeasures. Recent metagenomic surveys have uncovered an extensive diversity of HNVs and related paramyxoviruses circulating in wildlife, the majority of which remain uncharacterized due to the dearth of viral isolates. In lieu of viral isolates, reverse genetics systems offer an approach to derive infectious clones de novo in the laboratory, facilitating research into the biology, zoonotic potential, and pathogenicity of novel HNVs. This chapter explores the methodologies and applications of reverse genetics systems for novel HNVs, including considerations for virus sequence validation, full-length virus recovery, and the development of platforms such as minigenomes, replicons, and virus replicon particles. Such biologically-contained life cycle modeling systems enable research to be conducted at lower biocontainment, and provide accessible tools through which to investigate HNV biology. This work demonstrates the versatility of reverse genetics systems in advancing our understanding of high-consequence pathogens, enabling the proactive development of vaccines, antivirals, and diagnostic tools. By integrating these methodologies within a framework of biosafety and biosecurity, researchers can better prepare for and respond to future zoonotic threats.