Unveiling Intra-Clonal Diversity of Monkeypox Virus from Brazil’s First Outbreak Wave

The monkeypox virus (MPXV) is an emerging zoonotic pathogen responsible for mpox, a disease characterized by some smallpox-like symptoms, typically mild but occasionally fatal. The largest mpox recorded global outbreak began in May 2022, with over 162,000 cases across 140 countries. Herein, we have analyzed the intra-clonal diversity of MPXV obtained from a single skin lesion sample from a male patient (June 2022). Three viral clones were obtained following phenotypic evaluation of MPXV lysis plaque characteristics over a three-course infection in BSC-40 cells. Unlike the vaccinia virus Western Reserve (VACV-WR) strain, MPXV clones did not produce comet-like structures, suggesting reduced extracellular enveloped virus (EEV) morphotype release, which is associated with viral dissemination. Whole-genome sequencing and assembly identified subtle differences among clones. Comparative genomic analyses, including synteny and single nucleotide variation (SNV) calling, revealed intra-clonal differences and divergence from clade I and II references, although the variety of mutations found did not reveal possible variations at the protein level. Altogether, these findings suggest that although similar, it is possible that distinct MPXV variants may circulate together and can be found in a single exanthematous lesion.