Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses.

Henipaviruses, such as the prototypical Nipah virus and Hendra virus, are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, as well as for the emergent species, Ghana virus. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, application of this system to Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and identify two orally-efficacious antivirals effective against it. Further, we compare the compatibility of divergent henipavirus replicases with heterotypic viral genetic elements, providing valuable insights for how these species have evolved. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.