Detection of monkeypox virus with real-time PCR assays

Abstract

Background: Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak.

Objectives: We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak.

Study design: A TaqMan-based assay (E9L-NVAR) targets the orthopoxvirus DNA polymerase gene and detects Eurasian orthopoxviruses other than Variola. A hybridization assay, utilizing a MGB Eclipsetrade mark (Epoch Biosciences) probe, targets an envelope protein gene (B6R) and specifically detects monkeypox virus (MPXV). Assays were validated using coded orthopoxvirus DNA samples and used to evaluate lesion samples from five confirmed US monkeypox cases.

Results: E9L-NVAR did not detect variola (48 strains), North American orthopoxviruses (2), or DNA derived from non-poxviral rash illnesses. The assay reproducibly identified various concentrations of 13 Eurasian orthopoxvirus strains and was sensitive to 12.5 vaccinia genomes. The B6R assay recognized 15 different MPXV strains, while other orthopoxvirus (9) and bacteria (15) strains did not cross-react. Of the 13 human samples tested from confirmed cases, both assays identified 100% as containing MPXV DNA.

Conclusions: E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections.

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. The E9L-NVAR primers and probe are completely homologous with the vaccinia Copenhagen DNA sequence. The B6R primers and probe are completely homologous with the monkeypox (CB) DNA sequence. Virus strains: monkeypox (CB) MPXV-ZAI-1996-016 (Genbank AF380138); monkeypox (US) MPXV-USA-2003-039 (Genbank DQ011154); vaccinia Copenhagen (Genbank M35027); cowpox Brighton (Genbank AF482758); ectromelia Moscow (Genbank AF012825); camelpox Kazakhstan M-96 (Genbank AF438165); taterapox (Smith GL, personal communication); variola major Bangladesh (Genbank L22579); variola minor Garcia (Genbank Y16780). CB, Congo Basin; US, United States.