In recent years, the incidence, diversity, and geographical spread of MPXV have been continuously increasing. Additionally, there has been a shift from historically primarily zoonotic spill-over events with limited regional human-to-human transmission events to sustained and global human-to-human transmission. This was reported for the first time during the global outbreak in 2022, with retrospective evidence of ongoing human-to-human transmission of this clade in Nigeria dating back to 2016³². Since then, escalating human-to-human transmission has been seen for clade Ia in Kinshasa in 2024 (western DRC)¹³, and sustained human-to-human transmission has been documented for clade Ib in South Kivu (eastern DRC) since 2023¹². This clade Ib virus subsequently spread and resulted in sustained human-to-human transmission chains in Uganda and Burundi in 2024. In this study, we applied whole-genome sequencing of isolates collected from the first three months after the introduction of MPXV into Burundi to help understand the expansion and genomic diversity of the virus.

Based on the molecular clock analysis several different clusters could be identified. Assuming the diagnostic testing successfully detected the first cases and the virus was not present in the country before July 2024, there were at least three different introductions into the country, two of which mainly led to further spread of the virus within the country. This is in contrast with findings elsewhere where the clade Ib cases have been detected but have been contained efficiently with sometimes only a limited number of additional cases^33,34,35. Also in Burundi there have been sporadic cases without further spread, suggesting that the establishment of sustained human-to-human transmission is not purely a virus trait but is more dependent on high-risk behaviour patterns. Alternatively, the virus was already present in the country at an earlier time point but remained unnoticed. Although mpox surveillance was already implemented since October 2023 in Burundi it is a stigmatising disease and therefore the first introductions might have been missed.

Genomic rearrangement events have been reported for MPXV as a mechanism to compensate for comparatively low SNP rates of DNA viruses^36,37. A deletion event of approximately 1,140 bp in clade Ib viruses near the 5’ terminal region removes the D14L gene, one of five genes believed to be linked to increased virulence in clade Ia viruses³⁸. This gene is also missing in the clade II viruses^19,39. However, recently also a sexual transmission cluster of clade Ia has also been described⁴⁰ raising the question if clade Ia MPXV has the potential to be further spread in sexual networks mainly via sexual transmission. There was no indication of genomic rearrangement among the 98 clade Ib sequences analyzed in this study, and to the best of our knowledge, no published genomic rearrangements in MPXV clade Ib have been reported to date. This contrasts with genomic rearrangement events reported during the clade IIb outbreak, which ranged from 0.63% to 3.4%^10,36,37,38. However, further and longer sequencing efforts may be required to draw stronger conclusions.

A large proportion of the sequenced cases were adults above 20 years of age, representing 86,9% of the sequences. This does not necessarily reflect the current epidemiological situation in Burundi, where predominantly children have been infected with clade Ib MPXV with 39,5% of the cases aged below 15¹⁸. The reason for the bias is that only samples with enough sample volume were sequenced. However, we still think this sequence data is representative of the situation in the country in the first few months after the introduction of the virus because these adults can be used as a proxy for what is going on in the entire population since adults are most probably driving the transmission while children most probably acquire the infection via community transmission though direct contact with infected individuals.

There was one reported death among the 2447 confirmed clade Ib MPXV cases in Burundi (0,04%). This contrasts with the initial case fatality rates reported in the DRC with 1% for clade Ib in South Kivu province (670 cases) and 4.6% in the nationwide previously predominantly clade Ia outbreak^16,21. However, the case fatality rate is difficult to attribute in different settings, possibly due to age differences, overrepresentation of detections in hospitalized cases, surveillance coverage and/or co-morbidities. In the first three months of the outbreak in Burundi, mainly younger children were infected, which might account for this difference, although all cases investigated here were hospitalised.

In Burundi, after the initial introductions of MPXV the virus continued to spread within the country, mostly in an apparent monophyletic cluster, although Bayesian evolutionary analysis revealed that there were at least three different introductions with subsequent spread within the country. However, these results have to be interpreted with care, and more sequence data should be generated and shared from the current outbreak in neighbouring countries and the DRC. Likewise, some infections might have been missed, and we did not sequence all positive cases from Burundi, through which we also might have missed some different introductions into the country. Comparing our sequences to those publicly available, we noted the cases in Uganda are not linked to the cases detected in Burundi. However, more data are needed to draw any clear conclusions on this and to allow for further elucidation of potential between country transmission.

In conclusion, here we show the sustained circulation of the clade Ib MPXV in Burundi, most likely after initial introduction from the bordering province of South Kivu (DRC). The virus acquired several additional APOBEC-3 mediated mutations, in line with further ongoing human-to-human transmission^28,41. More genomic sequencing is needed from neighbouring countries and more recent time points, but it seems that the outbreak in Burundi is caused by in-country transmission after several different introductions of the virus into Burundi.