Monkeypox virus genome sequence from an imported case in Colombia

Katherine Laiton-Donato, Diego A. Álvarez-Díaz, Carlos Franco-Muñoz, Héctor A. Ruiz-Moreno, Paola Rojas-Estevez, Andres Prada, Alicia Rosales, Martha Lucia Ospina, Marcela Mercado-Reyes.

Authors details

Group Genomics of Emerging Microorganisms. Dirección de Investigación en Salud Pública and Dirección General, Instituto Nacional de Salud, Bogotá, Colombia.

Monkeypox virus (MPXV) is composed of a double-stranded DNA genome of approximately 197.209 bp. The current classification divides MPXV into two clades: Central African or Congo Basin clade (clade 1) and West African clade (clade 2 and clade 3). On June 28, 2022, 4357 cases were reported in 48 countries. Here we report a complete genome and phylogenetic analysis of a monkeypox case detected in Colombia.

Exudate from vesicular lesions was received on June 23, 2022, from a male patient with recent travel history to Spain. DNA purification was performed using 200 μl of sample and PureLink Viral RNA/DNA Mini Kit (Life Technologies, USA), according to the manufacturer’s instructions. DNA was quantified by fluorimetry with the Qubit dsDNA High Sensitivity Assay (Life Technologies, USA) on the Qubit 4.0 instrument (Life Technologies, USA). The metagenomics approach was performed using 400 ng of DNA using the native barcode kit EXP-NBD196 (Oxford Nanopore Technologies ONT, UK) and a 1:1 ratio of AMPure XP beads (Beckman Coulter, UK). The library was loaded onto FLO-MIN106 flow cells on the GridION sequencer (Oxford Nanopore Technologies ONT, UK).

Basecalling and demultiplexing were performed on Nanopore sequence reads using Guppy v.6.1.7 (Oxford Nanopore Technologies) and adapters were trimmed by using Porechop version 0.2.4. Processed reads were aligned against the Monkeypox virus reference genome (GenBank reference no. NC_063383.1) using minimap2 v.2.24. Variant calling for single-nucleotide variants was performed with Medaka v.1.15.0. Sites with depth less than 10x were masked with Ns. Maximum-likelihood phylogenetic reconstruction was performed on the alignment with 22 genomes, K3Pu+F+I nucleotide substitution model, and IQ-TREE software.

A total of 11951 reads mapped directly to a reference genome with 96.8% of coverage (190.898 bp) in the consensus sequence and submitted to the GISAID database. The sequence is available under the GISAID accession ID EPI_ISL_13511312

Phylogenetic analysis of the Colombian MPXV genome demonstrated its close relationship to the west African clade (clade 3) and closely related to genomes described during the 2022 outbreak (Figure 1).

Figure 1. Phylogenetic tree of Monkeypox virus. The tree was reconstructed by maximum likelihood with the estimated K3Pu+F+I nucleotide substitution model for the dataset of 23 genomes.

Declarations: This study was done according to the mandatory surveillance in public health of the National Institute of Health of Colombia.