title: Detection and typing of human pathogenic hantaviruses by real-time reverse transcription-PCR and pyrosequencing.
abstract: BACKGROUND
  Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens.
  
  
  METHODS
  We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing.
  
  
  RESULTS
  The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice.
  
  
  CONCLUSIONS
  The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.
authors: Institute of Virology, Helmut-Ruska-Haus, Charité Campus Mitte, Berlin, Germany.
authors: Marit
authors: M
authors: Kramski
authors: Helga
authors: H
authors: Meisel
authors: Boris
authors: B
authors: Klempa
authors: Detlev H
authors: DH
authors: Krüger
authors: Georg
authors: G
authors: Pauli
authors: Andreas
authors: A
authors: Nitsche
mesh_terms: D000818:Animals; D016679:Genome, Viral; D006476:Orthohantavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D018778:Hantavirus Infections / Q000821:virology; D006801:Humans; D051379:Mice; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
chemical_list: D012367:RNA, Viral