新規ヌクレアーゼ-多重置換増幅法によるサル痘ウイルスの効率的な全ゲノムシーケンシング

Monkeypox virus (MPXV) has a large double-stranded DNA genome (~200 kb), which presents challenges for whole-genome sequencing. Conventional enrichment methods have specific limitations. To address them, we developed a novel enrichment strategy combining nuclease treatment and multiple displacement amplification (MDA), along with terminal PCR to compensate for the reduced read depth at the genome termini. When applied to 18 historical isolates, this method yielded more than 96% MPXV-specific reads, enabled complete genome assembly, and demonstrated reproducibility and robustness in the phylogenetic analysis. It successfully sequenced low-titer samples (up to a Ct value of 33.5), suggesting a potential performance comparable to PCR-based methods, and demonstrated broad applicability to other Orthopoxviruses, such as Cowpox and Ectromelia viruses. The nuclease-MDA method is cost-effective, avoids the issue of primer/probe mismatches, and is applicable to low-titer samples. This strategy offers a competitive and alternative for MPXV and related Orthopoxviruses genomic surveillance and fundamental research.