Development of a replication-defective MPXV platform for fundamental and therapeutic research
Chen, J.; Hu, L.; Shi, N.; Tian, J.; Zhang, Y.; Tian, S.; Cao, X.; Ha, Z.; Cai, Q.; Lu, Y.; Smith, G. L.; Xie, Y.; Lu, H.; Zhang, P.; Zhang, R.
Show abstract
The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying monkeypox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a novel strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineered replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5L)--genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. Our work demonstrates the utility of replication-defective MPXV particles as a reliable platform for high-throughput antiviral discovery, offering significant advantages for both fundamental virology studies and therapeutic development against orthopoxviruses.
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