Kramski M, Meisel H, Klempa B, Krüger DH, Pauli G, Nitsche A (2007): Detection and typing of human pathogenic Hantaviruses by real-time RT-PCR and pyrosequencing
Clin. Chem. 53 (11): 1899-1905. Epub Aug 23.

Background: Human infections with hantaviruses commonly occur through inhalation of infectious, aerosolized excrements of rodents. Hantavirus-related diseases emerge as hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Since their clinical course can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is mainly based on serological assays. The detection of hantaviral RNA by the commonly used RT-PCR is difficult due to high sequence diversity of hantaviruses and low viral loads in clinical specimens.

Methods: In total, five real-time RT-PCR assays were developed. Three assays 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 for confirmation of the RT-PCR results or more detailed virus typing.

Results: The real-time RT-PCR assays presented were specific for the respective hantavirus species and optimized to run on two prominent real-time RT-PCR platforms, the LightCycler and the TaqMan 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 10–100 copies per reaction. With this assay, viral genome could be detected in 16/552 (2.5%) specimens of suspected hantavirus infections of human and mice.

Conclusions: The new assays are valuable for the detection, differentiation, and quantification of hantaviruses in clinical specimens from humans and from their natural hosts as well as for in vitro studies of hantaviruses.