A novel isothermal whole genome sequencing approach for Monkeypox Virus.

Abstract

Monkeypox virus (MPXV) is the zoonotic agent responsible for mpox, an often-self-limiting pox-like disease. Since May 2022, an outbreak characterized by increased human-to-human transmission was detected outside the endemic regions. Whole genome sequencing (WGS) has been successfully used to keep track of viral evolution during outbreaks or for surveillance of multiple pathogens of public health significance. Current WGS protocols for MPXV are either based on metagenomic sequencing or tiled-PCR amplification. The latter allows multiplexing due to the efficient enrichment of the viral DNA, however, mutations or the presence of different clades can negatively influence genome coverage yield. Here, we present the establishment of a novel isothermal WGS method for MPXV based on Phi29 DNA polymerase-based multiple displacement amplification (MDA) properties making use of only 6 primers. This approach yielded from 88% up to 100% genome coverage using either alkaline denatured extracted DNA or clinical material as starting material, with the highest coverage generated by clinical material. We demonstrate that this novel isothermal WGS protocol is suitable for monitoring viral evolution during MPXV outbreaks and surveillance in any conventional laboratory setting.