Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses

Abstract

Five of the known arenaviruses cause viral hemorrhagic fever in humans and are classified as biosafety level 4 pathogens. Four of the viruses, namely Junin, Guanarito, Machupo, and Sabia, belong to clade B of New World arenaviruses that also comprises the nonpathogenic viruses Tacaribe, Cupixi, and Amapari. To establish real-time reverse transcription (RT)-PCR assays for Junin and Guanarito virus based on fluorescence resonance energy transfer (FRET) probes, and a universal RT-PCR assay for all known clade B viruses with conventional read-out. Conserved sequences in the nucleoprotein gene were chosen as target sites for primers and FRET probes. A common set of primers was designed for all three assays. The assays were based on one-step RT-PCR reagents and were optimised with respect to analytical sensitivity using synthetic RNA templates. The real-time PCR assays detected about 0.5 and 5TCID(50) of cell culture-derived Junin and Guanarito virus, respectively. The universal clade B PCR amplified cell culture-derived RNA of Junin, Guanarito, Machupo, and Sabia virus (5-500TCID(50) per reaction), as well as RNA of Tacaribe, Cupixi, and Amapari virus. The PCR assays may be used as complementary diagnostic tests for pathogenic New World arenaviruses. The universal PCR assay could also be suitable for the detection of novel clade B arenaviruses in patients as well as in animal reservoirs.