APOBEC3-driven attenuation of the 2022 global outbreak monkeypox virus relative to its clade IIb ancestor

Monkeypox virus (MPXV) is a zoonotic virus endemic to Africa that has recently re-emerged, becoming the first orthopoxvirus with sustained human-to-human transmission since smallpox eradication. The 2022 global epidemic of MPXV was caused by Clade IIb virus (lineage B.1) that derived from the lineage A endemic to West Africa through APOBEC3 microevolution^1-3. Here, comparison of lineage B.1 virus with its closest earlier A.1 ancestor reveals that despite only 46 nucleotide differences, lineage B.1 has attenuating phenotypic changes. Both viruses replicated equivalently in cell culture, but B.1 had defects in long-range spread, which mapped to APOBEC3 mutation E353K in OPG057 (F13L). MPXV B.1 was also defective in innate immune control, inducing higher IFNβ and innate immune signalling gene expression, and showing reduced capacity to suppress interleukin-1β-mediated responses, which mapped to APOBEC3 mutation R48C in OPG047 (F3L). Finally, MPXV B.1 had reduced virulence in a mouse model of MPXV infection relative to the ancestral A.1 strain. Our work demonstrates that the 2022 global outbreak MPXV was intrinsically attenuated, with attenuating mutations that are absent in concurrent, more severe clade IIb outbreaks. Our data also reveal that APOBEC3 editing is a mechanism of MPXV phenotypic variation, potentially contributing to clade evolution and competition.