Tetracistronic minigenomes elucidate a functional promoter for Ghana virus and unveils Cedar virus replicase promiscuity for all henipaviruses

Batborne henipaviruses, such as Nipah and Hendra viruses, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often, the scope and capacity of research, which can be conducted at BSL-4, is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription, and replication-competent minigenomes for the Nipah, Hendra, and Cedar viruses, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficientlydriving replication and transcription of minigenomes from all tested henipaviruses. We also apply this technology to Ghana virus (GhV), an emergent species that has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. The use of our GhV system establishes the functionality of the GhV replicase and identifiestwo antivirals that are highly efficaciousagainst the GhV polymerase.