Monkeypox virus (MPXV) is a dangerous zoonotic pathogen that causes smallpox-like illness in monkeys. MPXV is classified into two phylogenetic groups: central African MPXV (endemic to the Congo Basin) and west African MPXV (endemic to West Africa). The two genotypes are associated with various disease severity and mortality rates: the western type is associated with milder disease and a lower fatality rate (Durski et al., 2018).

Despite being endemic in Sub-Saharan Africa, outbreaks have been linked to travellers coming back from endemic areas in the United States, the United Kingdom, and other countries (Hutson et al., 2007; Zumla et al., 2022; WHO, 2022).

Multiple cases of monkeypox are reported in various regions around the world since May 7th, 2022, including the United Kingdom, the United States, Australia, Canada and many European Union countries (Zumla et al., 2022). The West African MPXV genotype, according to current information, is fundamental to the outbreak (Isidro et al., 2022).

As part of the recent global outbreak, two complete MPXV genome samples were extracted in Slovenia.

Two cases of monkeypox came out in Slovenia on23rd May and 25th, 2022. Both cases were examined at the Department for Sexually Transmitted Diseases of Dermatovenerologic Clinic, University Medical Centre Ljubljana, after returning from the Canary Islands, Spain, for acute symptoms of orthopoxvirus infection, including general malaise, swollen inguinal nodes, and anogenital skin lesions.

Perianal swab samples and bioptic samples of perianally distributed papules were collected in UTM Viral Transport Media and tested positive for orthopoxviruses using 2 distinct real-time PCR assays (Schroeder et al. 2010 and RealStar Orthopoxvirus PCR Kit RUO, Altona, Hamburg, Germany). The West African genetic lineage of monkeypox virus (MPXV) was confirmed using MPXV-specific real-time PCR assays (Li et al. 2010). 

An initial draught genome sequence was obtained based on the nanopore long read maps of sample 1. Long reads were sequenced using an ONT GridION instrument with adaptive sequencing. The sequence MT903344.1 4 was used as a reference. For long-read mapping, Minimap2 (v2.20-r1061) (Li, 2018), samtools (v1.9) (Li et al., 2009), and iVar (v1.0) were used (Grubaugh et al., 2019). For further refinement, Medaka (v1.5.0) was used (Lee et al., 2021). Illumina reads (2 150 bp; 20 M paired reads) were captured for each sample using an Illumina NextSeq550 instrument and mapped to the draught genome sequence with bwa mem v0.7.17-r1188 (Li, 2013). 

The iVar-generated consensus sequences were refined using Pilon v1.23 (Walker et al., 2014). To generate preliminary functional annotations, Prokka v1.14.5 was used (Seeman, 2014). The entire MPXV genome sequences were submitted to GenBank under the accession numbers ON609725.2 6 (sample 1) and ON631241.1 2 (sample 2), with an average of 2380 and 4018 novel sequences covered.

GenBank and were used to obtain background and outbreak complete genome MPXV sequences from 2022. The substitution model GTR+F+R4 was used to infer a phylogenetic tree, and node support values were estimated using UFBootstrap (Katoh et al., 2013). Hoang and his colleagues (2018) The Slovenian sequences fit well within the outbreak’s phylogenetic cluster, confirming that the cases are molecularly related. 

Surprisingly, the first Belgian MPXV genome sequence from the 2022 outbreak (ITM-MPX-1-Belgium-2022) clustered with sequences from the previous outbreak rather than with the other outbreak sequences. This could be due to the bias from retaining reference sequence nucleotides in regions where sample sequence reads were not sufficiently covered during consensus calls.

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