THE FIRST HUMAN ISOLATE OF PUUMALA VIRUS IN SCANDINAVIA AS CULTURED FROM PHYTOHEMAGGLUTININ-STIMULATED LEUKOCYTES

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 i n the presence of phytohemagglutinin (PHA) in roller tubes during the first 72 hours of incubation followed by rolling culture for three wee ks in total. Thereafter the first subculture was done in a plastic fla sk and afterward at at least 6 week intervals. Antigen was first detec ted after 6 months and 2 weeks of culture. When tested by monoclonal a ntibodies and patient sera the isolate had the characteristics of a PU U virus. PCR amplification using PUU-specific primers and subsequent p artial sequencing of the S and M segments revealed that the Umea/305/h uman/95 virus differs from the Finnish PUU Sotkamo rodent prototype vi rus and is similar brat: not identical to rodent strains of PUU virus aquired from the same region as the patient isolate. it is we conclude d that the first human isolate of the etiologic agent of NE in Scandin avia was recovered from blood leucocytes stimulated with PHA by long-t erm culture in Vero E6 cells. The isolate belongs to the PUU serotype of hantaviruses as shown by its serologic profile and partial sequenci ng data. (C) 1997 Wiley-Liss, Inc.