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

J Clin Virol. 2005 Mar;32(3):229-35. doi: 10.1016/j.jcv.2004.07.011.

Abstract

Background: 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.

Objectives: 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.

Results: 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.

Conclusions: 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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arenaviridae Infections / diagnosis
  • Arenaviruses, New World / classification
  • Arenaviruses, New World / isolation & purification*
  • Chlorocebus aethiops
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Humans
  • Nucleoproteins / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Vero Cells
  • Viral Proteins / genetics

Substances

  • Fluorescent Dyes
  • Nucleoproteins
  • Viral Proteins