Identification of an extremely large-scale genomic rearrangement in the Mpox virus while retaining replicative capacity

Understanding the genomic diversity of the Mpox (formerly known as 'monkeypox') virus (MPXV) is important for monitoring viral evolution and dissemination. We encountered three clinical cases in our hospital during the outbreak period of Mpox in 2023 in Japan. The present study aims to report the largest genomic rearrangement observed to date in one of the clinical isolates and to demonstrate its viability in cell culture. Whole-genome sequencing and digital PCR were used to characterize the viral genomes. Viral isolation, microscopic observation and growth kinetics in Vero cells were performed to confirm viral replication. All three patients were men living with human immunodeficiency virus (HIV) and presented typical Mpox symptoms, such as rash, fever and pustules on the body surfaces, including near the genitals. Virus isolation was successful in all three cases. All viral strains belonged to clade IIb, lineage C.1. Notably, one strain exhibited a large-scale genomic rearrangement: a 5.5 kb deletion at the left variable region replaced by a 30.5 kb inverted sequence, the largest reported in clinical isolates. Despite this extensive genomic change, the strain maintained robust replication capacity and marked fusogenicity <i>in vitro</i>. We report, for the first time in clinical isolates, a massive genomic rearrangement in MPXV that does not impair viral replicability. This finding represents an example of genomic plasticity and provides a rare but noteworthy resource for future studies. Taken together, when performing genomic analyses of MPXV, aberrant genomic rearrangements should also be carefully considered alongside single-base substitutions.