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Microbiol. Dobrava, and Puumala viruses are known to cause HFRS in Russia, Asia, and Europe (22), whereas Sin Nombre and related viruses cause hantavirus pulmonary syndrome in the Americas [for Chlorthalidone reviews, see references 25 Chlorthalidone and 28). Each group of hantaviruses has a different rodent reservoir: Hantaan, Dobrava, and Seoul viruses are transmitted by rodents of the Murinae subfamily, Sin Nombre is transmitted by Sigmodontinae, and Puumala, Tula, Topografov, and Khabarovsk viruses are transmitted by Arvicolinae. Puumala virus, carried by the bank vole causes a relatively mild but invalidating form of HFRS (also called nephropathia epidemica) in northern and central Europe, particularly in Scandinavia and in the western parts of Russia (22, 23). Numerous cases are reported in Belgium, Germany, Austria, and in the Champagne-Ardennes and Franche-Comt foci in France (4, 7, 11, 12, 20; B. Le Guenno, M. A. Camprasse, J. C. Guilbaut, P. Lanoux, and B. Hoen, Letter, Lancet 343:114-115, 1994). Clinical manifestations are fever, conjunctival infections, thrombocytopenia, and transient renal failure. Detection of the viral genome by reverse transcription-PCR (RT-PCR) in blood or urine samples has been done largely (1, 10, 13, 31) because isolation of the virus in tissue culture is rarely successful. A rapid test by real-time RT-PCR was recently developed for Puumala virus (8). A sensitive immunoassay can also be used for the detection of viral antigens in human specimens (17). Although these techniques are useful to assess viremia, serological tests based on the detection of specific antibodies are widely used for routine diagnosis. During the acute phase of infection, the immunoglobulin M (IgM) level rises, followed by the production of IgG; the early antibody response is induced by nucleoprotein N, the major antigen (6, 18, 34, 40). Serological assays are based on viral antigens expressed in infected cells. However, massive production of viral proteins is rarely observed because the virus grows poorly in tissue culture. In addition, some hantaviruses must be manipulated in a high-security containment facility. Therefore, several laboratories have expressed the N protein as a recombinant protein in (6, 9, 24, 37) or in insect cells Chlorthalidone (5, 29, 30, 33, 36). In this study, we expressed the Chlorthalidone N protein of Puumala virus in mammalian cells via the Semliki Forest virus (SFV) replicon and compared its antigenic properties with those of the native antigen extracted from Puumala virus-infected cells. The recombinant antigen worked as well, or even better, than the native antigen, in the detection of IgM and IgG in patient sera by indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). It was also used to analyze sera or lung and kidney washes from wild bank voles and Chlorthalidone found very efficient for all these serological investigations. MATERIALS AND METHODS Cells. BHK-21 cells were grown in Glasgow minimal essential medium (MEM) supplemented with 5% fetal calf serum (FCS), 10% tryptose phosphate, and 10 mM HEPES. BSR cells (a clone of BHK-21) were cultured in Glasgow MEM supplemented with 10% FCS, and TRAIL-R2 Vero E6 cells were grown in Dulbecco modified Eagle medium supplemented with 5% FCS. Penicillin (5 U/ml) and streptomycin (5 g/ml) were added. The cells were incubated at 37C in a 5% CO2 atmosphere. Virus and native antigen for ELISA. Stocks of Puumala virus (strain Cg 13891) were produced by infecting semiconfluent Vero E6 cells at a low multiplicity of infection (MOI) of 10?3 to 10?4. To produce Puumala virus antigen for ELISA, Vero E6 cells were infected and incubated for approximately 2 weeks. The infected Vero E6 cells were trypsinized and mixed with an equal number of fresh cells, which were then used to seed new flasks. When 80% of the cells gave positive results in an IFA, which usually required three passages, the cells were collected, washed with borate buffer (50 mM sodium borate [pH 9.0], 120 mM NaCl), and centrifuged at a low speed. The cell pellet was suspended in 10 volumes of borate buffer containing 1% Triton X-100. Cell lysis was achieved by sonication (four 30-s pulses) at maximal power (300 W). Cell debris was centrifuged at 6,000 polymerase (Eurogentec) according to the manufacturer’s instructions. Plasmids were constructed by standard protocols (2). The PCR product was digested by is the recognized reservoir of Puumala.