The first human isolate of Puumala virus in Scandinavia as cultured from phytohemagglutinin stimulated leucocytes.

A virus isolate was recovered from blood leucocytes of a patient with nephropathia epidemica (NE). Leucocytes were isolated from EDTA-blood by dextran sedimentation and cultured on monolayers of Vero E6 cells in the presence of phytohemagglutinin (PHA) in roller tubes during the first 72 hours of incubation followed by rolling culture for three weeks in total. Thereafter the first subculture was done in a plastic flask and afterward at at least 6 week intervals. Antigen was first detected after 6 months and 2 weeks of culture. When tested by monoclonal antibodies and patient sera the isolate had the characteristics of a PUU virus. PCR amplification using PUU-specific primers and subsequent partial sequencing of the S and M segments revealed that the Umeå/305/human/95 virus differs from the Finnish PUU Sotkamo rodent prototype virus and is similar but not identical to rodent strains of PUU virus acquired from the same region as the patient isolate. It is we concluded that the first human isolate of the etiologic agent of NE in Scandinavia was recovered from blood leucocytes stimulated with PHA by long-term culture in Vero E6 cells. The isolate belongs to the PUU serotype of hantaviruses as shown by its serologic profile and partial sequencing data.