Detection and typing of human pathogenic hantaviruses by real-time reverse transcription-PCR and pyrosequencing

Background: Because the clinical course of human in-fections 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 tran-scription (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 hantavi-ruses 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 wa