Abstract Since the Mpox virus (MPXV) outbreak in 2022, there have been 97,745 cases and 203 fatalities. This outbreak features increased transmission efficiency and a higher infection rate in the MSM population, with the underlying causes remaining unknown. The requirement of BSL-3 laboratories poses a barrier to MPXV research and antiviral development. Here, we report an efficient and safe trans-complementary system that produces a single-round infectious MPXV, preserving the virus’s authentic architecture and enabling it to complete its life cycle in complementing cells. This deficient MPXV genome, lacking two essential genes crucial for late gene transcription and viral assembly, is restricted to a single-round infection in non-complementing cell lines. Notably, SCID mice inoculated with this deficient MPXV exhibited no detectable disease or viral load. This deficient MPXV platform has demonstrated its capacity to study innate immunity and cell death during infection in complementing cells. It can also be used for antibody neutralization assays and anti-MPXV drug evaluation. This trans-complementation platform, safe for use in low-biosafety laboratories, offers a valuable resource for MPXV research and countermeasure development.

An Efficient and Safe Trans-complementation System of Mpox Virus Reproduces Authentic Viral Architecture and Infection