Detection and subsequent sequencing of Puumala virus from human specimens by PCR

Abstract

A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.