LAUSR

New York City (NYC) was a major hotspot during the 2022 multi-country Mpox virus (MPXV) outbreak in the U.S. To investigate the genomic characteristics of MPXV in NYC, we sequenced 1138 specimens from 758 individuals using PrimalSeq and Illumina technology and performed phylogenomic analyses alongside 2967 global MPXV sequences. Nextclade lineage assignment revealed a NYC-specific B.1.12 lineage, with phylogenetic analysis showing unique clusters in NYC and North America. MPXV lineage B-specific mutations were predominantly driven by APOBEC3 activity (53/58 mutations). APOBEC3-associated mutations were significantly higher in 2022 outbreak sequences compared to those before 2022. Intra-host genome diversity was investigated in individuals with multiple specimens (n = 360). This analysis showed that 6.4% of these individuals had distinct genomic profiles, with 4.2% likely due to co-infections with distinct MPXV strains. This study identified unique MPXV genomic profiles in NYC, estimated concurrent infections with multiple MPXV strains and emphasized the need for enhanced surveillance and public health strategies to address co-infections, especially through collaboration with labs, clinics, and community groups. Using MPXV genomes specific to New York City, phylogenetic clusters were identified. Most mutations were driven by ABOBEC3. The prevalence of coinfections with distinct strains was ~4.2% and results may improve MPXV genomic epidemiology applications.