In July 2024 Uganda reported initial human mpox cases. Between July and December 2024, investigators collected 1,072 samples from persons with clinically suspected mpox to characterize circulating viral agents. By May 28, 2025, national reports cited up to 6,479 confirmed mpox cases with 44 deaths; despite that, many clinically suspected mpox patients tested negative by MPXV real-time PCR. Because early rash stages of mpox can resemble other exanthematous illnesses, the study aimed to use genomic methods to refine differential diagnoses and to characterize circulating monkeypox virus and other pathogens.
Real-time PCR was performed on all 1,072 samples. Of these, 193 samples were MPXV PCR–positive. Researchers genotyped 115 MPXV-positive specimens using target-enrichment next-generation sequencing (NGS). From the 879 MPXV-PCR–negative samples, a randomly selected subset of 127 (14% of negatives) underwent metagenomic NGS and target-enrichment NGS, with MiSeq sequencing platforms used for data generation. Analysis pipelines included an in-house metagenomic pipeline from UVRI, Abbott’s DiVir pipeline, and One Codex software. Phylogenetic reconstructions used IQ-TREE and Squirrel software for mutational mapping.
Among the 193 confirmed mpox patients, 48.7% were male and 51.3% female. Nearly half of confirmed cases (47.8%) occurred in persons aged 16–30 years. Approximately 65% of the study samples were collected from central Uganda. The article provides additional demographic breakdowns and tables in Appendix 1.
Phylogenetic analysis of 115 newly generated MPXV genomes showed that the Ugandan sequences belonged to clade 1b. These new sequences clustered closely with previously reported sequences from Uganda and the Democratic Republic of Congo, indicating genetic relatedness to regional clade 1b strains. Although related, the mutational profiles of newly sequenced strains displayed minimal overlap with earlier Uganda sequences, suggesting ongoing divergence within the clade.
Mutational mapping performed on 137 Uganda MPXV genomes (115 new and 22 previously reported) relative to a Zaire-1979 reference identified 284 mutation events, inclusive of synonymous, nonsynonymous, and intergenic changes. Of these 284 events, 118 (41.4%) were consistent with APOBEC3-mediated cytosine deamination, identified as TC->TT or GA->AA dinucleotide substitutions. Squirrel software was used to infer ancestral states and to map APOBEC and non-APOBEC mutations onto phylogenetic branches.
Functional annotation focusing on nonsynonymous changes indicated that recent Uganda strains acquired amino acid substitutions in proteins linked to inhibition of host responses (including inflammasome components and T-cell activation pathways), immune evasion, transcription and RNA metabolism (for example, poly-A polymerase and DNA-dependent RNA polymerase), virion morphogenesis, and viral entry/egress. The distribution of affected genes differed between sequences from this study and previously reported Uganda sequences; previous strains showed fewer annotations related to apoptosis inhibition, viral maturation, and mRNA (de)capping than strains sequenced in this investigation.
Sequencing of the 127 MPXV-PCR–negative samples detected varicella zoster virus (VZV) with >80% genome coverage in 106 specimens (83%). Phylogenetic reconstruction of 106 high-coverage VZV genomes generated in this study, together with publicly available complete genomes, showed that most VZV sequences from this investigation formed a single well-supported Uganda clade. These Uganda VZV sequences were genetically similar to sequences previously identified in Nigeria, Ghana, and Guinea-Bissau. A small number of study sequences resolved outside the predominant Uganda clade and formed two distinct lineages.
Beyond VZV, metagenomic sequencing identified additional viral and bacterial agents in MPXV-negative specimens. Coxsackievirus A6 was detected in 9 samples (7%), measles virus genotype B3 in 7 samples (6%), and various polyomaviruses in 5 samples (4%). Bacterial organisms identified included Cutibacterium acnes, Staphylococcus aureus, Streptococcus pyogenes, and Corynebacterium diphtheriae. The proportions above reflect numbers reported for the sequenced negative subset and were determined based on genome coverage and pipeline detection thresholds.
The authors submitted 115 MPXV sequences to GISAID under EpiPox in nonsequential installments and deposited VZV genomes to GenBank (BioProject no. PRJNA1281018; accession nos. provided in the paper). The study highlights that clinically suspected mpox cases may include other pathogens, particularly varicella zoster virus, and demonstrates that molecular diagnostics and genomic surveillance can refine differential diagnosis. The genomic findings—clade 1b MPXV circulation and APOBEC3-associated mutational patterns—underscore the value of continued sequencing to monitor viral evolution and to strengthen surveillance of co-circulating rash illnesses.
Note: Additional tabular details, accession numbers, and full mutational tables are reported in the article appendices and public sequence repositories as cited by the authors.