During 2022, an unprecedented multicountry outbreak of monkeypox virus (MPXV) infection among humans was detected. The first verified mpox cases in Europe were reported in mid-May 2022 with no apparent link to MPXV-endemic countries, but patients shared travel history to Lisbon, Portugal, and Gran Canaria, Canary Islands, as well as sexual behavior (men who have sex with men [MSM]) (1). The first draft sequence of the outbreak-related genome from Portugal was published on May 19, 2022 (J. Isidro, unpub. data, https://virological.org/t/first-draft-genome-sequence-of-monkeypox-virus-associated-with-the-suspected-multi-country-outbreak-may-2022-confirmed-case-in-portugal/799). During the following weeks, several closely related MPXV genomes were reported from other countries in Europe, resulting from travel-associated and community-transmitted infections. The clinical picture of those infections (anogenital lesions or rash and enlarged inguinal lymph nodes) (2), together with the epidemiologic data, suggested human-to-human transmission by sexual contact, mainly among MSM (3); however, other routes of transmission may also have played roles (4). As the number of verified mpox cases increased, on July 23, 2022, the World Health Organization declared MPXV a Public Health Emergency of International Concern (https://www.who.int/director-general/speeches), although the epidemic has since waned. We describe the molecular and clinical characteristics of MPXV introduced to Finland during late May–early June 2022 (Table). The patients provided written informed consent for use of their case details and medical images in this study.

We investigated 4 patients who exhibited systemic mpox symptoms, such as fever and skin lesions (Appendix). The patients were epidemiologically unrelated to each other; however, all reported travel in southern Europe, declared themselves to be MSM, and declared recent unprotected sexual exposure with previously unknown partners (Table). Two patients were HIV positive. Orthopoxvirus real-time PCR of individual skin lesion samples detected orthopoxvirus, which was later verified as MPXV by hemagglutinin gene sequencing with MinION (Oxford Nanopore Technologies, https://nanoporetech.com) (Appendix). The sample from patient 1 was also sequenced by MinION and on May 27, 2022, produced an MPXV draft genome. The whole genomes of all samples were subsequently sequenced by using Illumina NovaSeq (https://www.illumina.com). As of November 8, 2022, the total number of verified cases in Finland reached 42, but no further virus transmission from those patients has been reported.

Figure

Figure. Phylogenetic tree of monkeypox virus (MPXV) sequences used in study of intrahost viral genome variation in patient with early monkeypox virus infection, Finland, 2022. The tree was inferred by the...

We obtained complete MPXV genomes from 3 of the 4 patients: patient 1 (penis, quantitative cycle [Cq] 19.77), patient 2 (face, Cq 26.29), and patient 4 (perianal skin, Cq 23.4); we could obtain only a fragmental genome from patient 3 (hand, Cq 33.38). In the phylogenetic analysis, the consensus sequence of MPXV genome from patient 1 (GenBank accession no. ON782021) clustered with lineage B.1.3 genomes (Figure). The members of that cluster share 3 substitutions: nonsynonymous G55133A (R665C in OPG074 protein), synonymous C64426T, and nonsynonymous G190660A (R84K in NTB03_gb174 protein), according to National Center for Biotechnology Information reference sequence NC_063383 coordinates (equivalent to the mutations addressed as G55142A, C64435T, G190675A [5]). The sequence from patient 2 (GenBank accession no. ON782022) was identical to the early sequences first detected in Portugal (6) and thereafter in various other countries. The sequence from patient 4 (GenBank accession no. ON959143) had 4 nt substitutions: C89906T (OPG110: S92F), G94798A (OPG115: E47K), C150831T, and C188491T. Two of those sequences (C89906T and G94798A) were shared with genomes from the United Kingdom, Portugal, Spain, and Germany.

The 3 nt substitutions detected in the patient 1 sequence were not fixed but rather contained minority variants with the frequencies of 10% (G55133A, depth 2231; nucleotide counts G = 233, A = 1997), 12% (C64426T, depth 2685; C = 308, T = 2364), and 13% (G190660A, depth 2685; G = 280, A = 1872). On the other hand, in the members of the same clade from Slovenia (GenBank accession no. ON609725) and France (GenBank accession no. ON622722), all 3 mutations were fixed (allele frequency >99.7%). The mutational signature of the major and minor intralesion single-nucleotide variant (SNV) findings in patient 1 is consistent with the effects of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) enzyme, which has been suggested to drive the CT>TT and GA>AA conversions in recent MPXV evolution (6; A. O’Toole et al., unpub. data, https://virological.org/t/initial-observations-about-putative-apobec3-deaminase-editing-driving-short-term-evolution-of-mpxv-since-2017/830). A similar phenomenon, the fixation of minor intralesion SNVs along the transmission chain, was observed in 5 of the 15 samples from Portugal sequenced in May 2022 (6) and in a publicly available MPXV sequence dataset (A. Nekrutenko et al., unpub. data, https://virological.org/t/mpxv-intrahost-variation-in-the-context-of-apobec-deamination-an-initial-look/856), suggesting that this pattern might be a general pattern of evolution for the 2022 MPXV outbreak. However, in contrast to the previous findings (6; A. Nekrutenko et al., unpub. data, https://virological.org/t/mpxv-intrahost-variation-in-the-context-of-apobec-deamination-an-initial-look/856), both the major and minor SNV genotypes from patient 1 could be found fixed in previously reported MPXV sequences.

In conclusion, we demonstrate intrahost MPXV variation within a single lesion from one of the patients with infection introduced to Finland. Most of the sequence reads in that sample contained APOBEC3-related mutations, which may have emerged from the ancestral minor variant present in this sample. However, because the majority and minority nucleotides in that sample are also found fixed in sequences from other countries, we cannot resolve whether this observation relates to contemporary APOBEC3-driven evolution or to co-infection.

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