Detection of nephropathia epidemica virus RNA in patient samples using a nested primer-based polymerase chain reaction

A nested primer-based polymerase chain reaction was constructed for the detection of Puumala virus RNA in patient samples. Puumala virus RNA was detected in cells from the urinary and the respiratory tracts and in peripheral blood mononuclear cells of patients with nephropathia epidemica. After inoculation with nephropathia epidemica patient material on Vero E6 cells and propagation for nine passages (4 months), Puumala virus RNA was detected at every passage. Hybridization under high-stringency conditions revealed that the overall nucleotide homology between the different patient isolates and the prototype strain (Puumala) is high. Using cDNA from Hallnas B1 strain as a probe, hybridization occurred only under low-stringency conditions.