Objective To establish a whole-genome sequencing (WGS) workflow and molecular tracing method for monkeypox virus (MpxV) based on amplicon sequencing technology combined with a high-throughput sequencing platform and bioinformatics analysis techniques, providing technical support for molecular epidemiological research of monkeypox cases in Dalian city.

Methods By taking total nucleic acid extracted from vesicle fluid samples of monkeypox cases as templates, a cDNA library was constructed on the Ion Torrent sequencing platform via WGS technology. Template preparation and sequencing were completed. Using the built-in plug-ins of Ion GeneStudio S5 Plus and NC063383.1 as the reference sequence, data quality control, base identification, sequence alignment, variant analysis, and whole-genome sequence assembly were performed to obtain consensus sequences (Fasta files). Online analysis tools were used to analyze the whole-genome characteristics, genotyping, and genetic evolutionary relationships of MpxV.

Results The cycle threshold (Ct) values of MpxV nucleic acid detection in vesicle fluid samples from the two patients were 28.03 and 29.60, respectively. The consensus sequence obtained from sample 1 was a 197 084 bp MpxV whole-genome sequence with a total of 3 470 722 reads, an average sequencing depth of 3 205, an average read length of 182 bp, and approximately 98.8% coverage. The consensus sequence obtained from sample 2 was a 197 099 bp MpxV whole-genome sequence with a total of 1 440 296 reads, an average sequencing depth of 746.7, an average read length of 105 bp, and approximately 97.8% coverage. Sample 1 had 84 single nucleotide mutations and 40 amino acid mutations, while sample 2 had 85 single nucleotide mutations and 42 amino acid mutations. Both samples belonged to the C.1.1 sub-branch of the MpxV IIb clade (West African clade). hMpxV/China/ DLCDC-01 was closely related to the sequences EPI_ISL18213375, EPI_ISL19058867, EPI_ISL18213374, EPI_ISL19058866, and EPI_ISL19058873 submitted from Guangdong province, China, and EPIISL_18059184 submitted from Yunnan province, China. hMpxV/China/DLCDC-02 was not homologous to hMpxV/China/DLCDC-01 or the 20 valid sequences from China uploaded to GISAID.

Conclusions Consensus sequences of MpxV were obtained from vesicle fluid samples of two patients, successfully establishing WGS technology, bioinformatics analysis, and molecular tracing methods for MpxV in Dalian city.