B-cell epitopes in the nucleocapsid protein (N) of Puumala (PUU) virus were investigated by use of truncated recombinant proteins and overlapping peptides. Six of seven epitopes, recognized by bank vole monoclonal antibodies, were localized within the amino-terminal region of the protein (aa 1-79). Polyclonal antibodies from wild-trapped or experimentally infected bank voles identified epitopes located over the entire protein. Antibody end-point titers to different N fragments indicated that the amino-terminal region is the major antigenic target in PUU virus-infected bank voles. To investigate the role of PUU virus N in protective immunity, we analyzed the immunogenicity of truncated recombinant N and developed an animal model based on colonized bank voles. No PUU virus N antigen, nor any glycoprotein-specific antibodies, could be detected after virus challenge in animals immunized with an amino-terminal fragment (aa 1-118), a fragment covering two thirds of the animals immunized with shorter N fragments displayed either N antigen, or glycoprotein-specific antibodies, suggestive of partial protection. Prechallenge sera from all groups of immunized animals were found negative or only weakly positive for neutralizing antibodies when assayed by focus reduction neutralization test, which indicated an important role for cell-mediated immunity in protection.

In order to investigate rodent host specificity of European hantaviruses, experimental infection of colonized and wild-trapped rodents was performed. In addition to the natural rodent reservoir, Clethrionomys glareolus, Puumala hantavirus (PUUV) could infect colonized Microtus agrestis and Lemmus sibiricus, but not Syrian hamsters or Balb/C mice. Neither C. glareolus, nor M. agrestis, could be readily infected by Tula hantavirus (TULV). Wild-trapped Apodemus flavicollis and A. agrarius, the natural reservoirs of Dobrava (DOBV) and Saaremaa (SAAV) hantaviruses, respectively, could both be infected by SAAV. NMRI mice could also be infected by SAAV, but with lower efficiency as compared to Apodemus mice. Balb/C and NMRI laboratory mice, but not C. glareolus, could be infected by DOBV. To our knowledge, this is the first time DOBV and SAAV have been shown to infect adult laboratory mice. Moreover, potential hantavirus spillover infections were investigated in wild-trapped rodents. In addition to the natural host C. glareolus, we also found M. arvalis and A. sylvaticus with a history of PUUV infection. We did not find any C. glareolus or A. sylvaticus infected with TULV, a hantavirus which is known to circulate in the same geographical regions of Belgium.

Tick-borne encephalitis virus (TBEV) is a severe problem in Estonia. In the present article the first genetic analysis of Estonian TBEV strains is described. In total, seven TBEV strains were isolated from ticks (Ixodes ricinus and I. persulcaus), rodents (Apodemus agrarius and Cletrionomys glareolus), and serum from a tick-borne encephalitis (TBE) patient. The nucleic acid sequences of the viral genome encoding almost the complete E protein (nt 41-1250) and the 3 0 -NCR-termini of the Estonian TBEV strains were determined by direct sequencing of RT-PCR products. The results showed that all three known TBEV subtypes, Western TBEV (W-TBEV), Far-Eastern TBEV (FE-TBEV), and Siberian TBEV (S-TBEV), co-circulate in Estonia. The Estonian TBEV strains of the S-TBEV and W-TBEV subtypes clustered with the previously reported strains from Latvia and Lithuania. Within the FE-TBEV subtype, however, the Estonian strain Est2546 clustered together with the strain Sofjin, originating from the Far-East of Russia, but not with the strain RK1424, isolated in the neighboring Latvia. This suggests a different evolutionary history for the Estonian and the Latvian strains in the FE-TBEV subtype. The Estonian TBEV strain (Est3535), which belonged to the S-TBEV subtype, had an organization of the 3 0 -NCR similar to that of strains from the Far-East of Russia (Irkutsk). The 3 0 -NCRs of Estonian strains of the W-TBEV subtype (Est3051, Est3053, Est3476, and Est3509) were very similar to those of the strain Ljubljana I from the Balkans. In the 3 0 -NCR sequence of the Estonian strain Est2546, which belonged to the FE-TBEV subtype, a deletion from position 10461 to 10810 extending approximately 10 nucleotides into the core element, was detected.

This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.