Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA

Abstract

Orthopoxvirus monkeypox (MPXV) forms two distinct clades: the MPXV Congo Basin clade viruses are endemic in the Congo Basin, human illness typically presents with symptoms similar to discrete, ordinary smallpox and has a case fatality rate of approximately 10% in unvaccinated populations; the MPXV West African clade viruses have been isolated in West Africa and appear to cause a less severe, and less inter-human transmissible disease. Recently, monkeypox outbreaks were reported in US and Sudan caused by MPXV West African and Congo Basin strains respectively. These events demonstrated the ability and trend of the virus to exploit new hosts and emerge globally; it also emphasizes the need for the diagnosis of MPXV, especially the ability to distinguish between Congo Basin and West African monkeypox strains. In this study, three new real-time PCR assays based on TaqMan probe technology were reported: the MPXV West African specific, Congo Basin strain specific and MPXV generic assays. The new assays demonstrated good specificity and sensitivity in the validation study with multiple platforms and various PCR reagent kits, and will improve the rapid detection and differentiation of monkeypox infections from other rash illness.

Fig. 1

Alignment of primers and probes with orthopoxviral DNA. The primers and probe for each of the real-time PCR assays are aligned with the targeted sequence of DNA within several orthopoxviral species. Virus strains: monkeypox West African strains MPXV_USA03, MPXV_SLE; monkeypox Congo Basin strains MPXV-ZAI79, MPXV_RCG03; variola major VARV_BGD75, VARV_CHN48; variola minor VARV_BRA66; camelpox CMLV_CMS, vaccinia VACV_WR, VACV_Acam2, VACV_DUKE; cowpox CPXV_GRI, CPXV_GER91; ectromelia Moscow ECTV_MOS. (A) The probe sequence of West African MPXV_G2R_WA assay has three bases deletion comparing to that of Congo Basin strains. (B) The MPXV West African strain lost complement binding protein C3L gene and are not in the sequences alignments. (C) The MPXV generic assay primers and probe are completely homologous within the MPXV DNA sequence except one SNPs in forward primer sequence.