Molecular cloning of a monkeypox virus clade IIb genome and construction of an attenuated mutant for basic and translational research

Monkeypox virus (MPXV) is a rodent-borne orthopoxvirus causing mpox disease upon zoonotic transmission to humans. Recent global outbreaks have raised awareness and highlighted the need for basic and translational research. However, research is hampered by the classification of MPXV as a Risk Group 3 pathogen. In an effort to overcome this limitation, we generated a complete full-length MPXV clade IIb bacterial artificial chromosome (BAC) clone by single-step transformation-associated recombination (STAR) cloning in yeast. The integrity of the cloned MPXV genome sequence and the replication capability of the BAC-derived virus were verified. To obtain an attenuated mutant, the viral thymidine kinase (TK) gene, OPG101, was deleted by BAC recombineering. The TK-deficient MPXV mutant replicated to similar titers as the wild-type virus in cell culture but was attenuated in vivo, as shown in the CAST/EiJ mouse intradermal infection model. The attenuated TK-deficient MPXV mutant has the potential to be classified as a Risk Group 2 pathogen by regulatory authorities. This would allow research to be conducted in Biosafety Level 2 laboratories and greatly facilitate basic and applied MPXV research. For example, we demonstrate that the TK-deficient virus can be used for the detection of neutralizing antibodies in human serum samples or for antiviral drug testing.