Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool

Abstract

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), micro -capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

FIG. 1.

Western blot analysis of recombinant Puumala virus nucleoprotein. Proteins from 4 × 10⁴ BSR cells infected with SFV-N_Puu, SFV-1, or Vero E6 cells not infected (mock) or infected with Puumala virus (Puumala V) or Hantaan virus (HTN V) were analyzed in a 10% polyacrylamide gel, transferred to a membrane incubated with hamster anti-Puumala antibodies revealed with peroxidase-labeled secondary antibodies. The positions of N proteins from Puumala and Hantaan virus (N Puu and N HTN, respectively) are indicated to the right of the blot. The positions of molecular size standards (in kilodaltons) are indicated to the left of the blot.

FIG. 2.

Immunofluorescence of the recombinant Puumala virus nucleoprotein expression. (A) BHK cells infected with SFV-N (MOI = 0.1), (B and C) BSR cells infected with SFV-N (MOI = 3) or not infected (C). Cells were fixed in situ with methanol and analyzed using hamster serum raised against Puumula virus, followed by a fluorescein-labeled secondary antibody.

FIG. 3.

Titration curves of IgM and IgG in HFRS patient sera by ELISA. ELISA for IgM (A) and IgG (B) detection in human sera using the recombinant (♦) or native (▴) antigen.

FIG. 4.

Titration curve of IgG in wild rodent samples by ELISA. ELISA for IgG detection in a pool of wild rodent samples using recombinant nucleoprotein (♦) and native antigen (▴).