Abstract

Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak.We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak.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.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.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.