High-resolution single nucleotide polymorphisms detect chronological recombination events during Mpox Ib outbreak

Viral recombination can rapidly generate new variants and influence outbreak severity. Currently, a method to track viral recombination events at high genomic resolution during natural transmission remains unavailable. Here we describe a novel linkage disequilibrium method and the first report of viral recombination events over time by using single nucleotide polymorphisms (SNPs) as natural genetic markers. Combined with phylogenetic data, we analyzed mpox virus (MPXV) Ib genome during 2023-2025 outbreak and found that 71 recombination events among Ib genomes, yielding a recombination frequency (Rf) and rate of 0.16%/kbp and 0.11%/kbp/year, respectively. Phylogenetic time analysis also shows that recombination began as early as December 23, 2023. Rf of MPXV Ib is 8.8-fold lower compared to vaccinia virus (1.5%/kbp) in vitro. Recombination and linkage hotspots are enriched at both inverted terminal repeat and variable regions of MPXV Ib genome. Our data not only reports the first Rf and rate of natural human transmission in the poxvirus family but also provides a powerful genomic surveillance tool for better understanding transmission and evolution of any viral outbreak globally.