Serological divergence of Dobrava and Saaremaa hantaviruses: evidence for two distinct serotypes.

In order to investigate the serological relationship of Dobrava hantavirus (DOBV, originating from Slovenia) and the Dobrava-like Saaremaa virus (SAAV, recently discovered in Estonia) we analysed 37 human serum samples, 24 from Estonia and 13 from the Balkans, by focus reduction neutralization test (FRNT). Most of the Estonian sera (19), including all sera from Saaremaa island (12), reacted with higher FRNT end-point titres to the local SAAV; the majority of them (15 and 11, respectively), with at least fourfold or higher titres to SAAV than to DOBV. In contrast, out of the 13 sera collected in Slovenia, Bosnia-Herzegovina and Greece, only one reacted more strongly with SAAV (with a twofold higher titre), while 10 of these sera reacted more strongly with the local DOBV (9/10 with fourfold or higher titres). These results indicate that DOBV and SAAV define unique hantavirus serotypes.

Modifying the cellular transport of DNA-based vaccines alters the immune response to hantavirus nucleocapsid protein.

Puumala virus is a member of the hantavirus genus (family Bunyaviridae) and is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) in Europe. A genetic vaccination approach was conducted to investigate if the immune response could be modulated using different cellular secretion and/or localisation signals, and the immune responses were analysed in BALB/c mice and in a bank vole infectious model. Rodents vaccinated with DNA constructs encoding the antigen fused to an amino-terminal secretion signal raised significantly higher antibody levels when compared to using constructs lacking secretion signals. Furthermore, the ratios of the IgG subclasses (IgG2a/IgG1) were raised by the use of cellular localisation signals, indicating a more pronounced Th1-type of immune response. The majority of the mice, or bank voles, immunised with DNA encoding a secreted form of the antigen showed a positive lymphoproliferative response and were protected against challenge with Puumala virus (strain Kazan-wt).

Puumala (PUU) hantavirus strain differences and insertion positions in the hepatitis B virus core antigen influence B-cell immunogenicity and protective potential of core-derived particles.

Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1-45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1-45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40-50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18-20 N (aa 1-45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75-119 of PUU (CG18-20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231-240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge.

A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus.

A combinatorial human antibody Fab pComb3H library, generated from splenic lymphocytes of a Puumala hantavirus (PUUV) immune individual, was selected against PUUV using the phage display technique. Panning was carried out with antigens immobilized by MAbs directed to the two PUUV envelope glycoproteins G1 and G2. Thirteen Fabs, with reactivity directed to PUUV and specifically the G2 protein, as assessed by immunofluorescence and ELISA respectively, were isolated in crude preparations. By a focus reduction neutralization test (FRNT), four of the 13 crude Fab preparations exhibited type-specific neutralization of PUUV (strain Sotkamo) with 44-54% reduction in the number of foci. After affinity purification, the four Fab clones exhibited 50% focus reduction of PUUV at concentrations below 2 microg/ml. Sequencing of the heavy and light chain complementarity determining regions (CDR) 1-3 showed that the four selected clones were identical within the antibody binding regions. In inhibition tests with the PUUV G2-specific MAbs, 4G2 and 1C9, a new epitope important for neutralization, designated as G2-a3, was defined. This epitope, overlapping partially the neutralizing epitope recognized by the human MAb 1C9, seems to be unique for the PUUV serotype since none of the Fab clones neutralized any of the other hantaviruses tested.

Dobrava virus infection: serological diagnosis and cross-reactions to other hantaviruses.

Recent data have shown that Dobrava (DOB) hantavirus is the cause of severe haemorrhagic fever with renal syndrome (HFRS) in central and eastern Europe. To determine whether serological assays need to be based on the homologous viral antigen rather than on closely related hantavirus antigens, acute and convalescent sera from patients with HFRS collected in former Yugoslavia were examined for IgM and IgG to three hantavirus antigens; DOB, Hantaan (HTN) and Puumala (PUU). Focus reduction neutralization test was included for comparison and confirmation of the enzyme-linked immunosorbent assay (ELISA) results. Although the results showed that the cross-reactivity was high between these three antigens during the acute phase of the disease, one of 155 patients serum samples reacted only in the DOB antigen-based IgM assay. The evaluation of IgG reactivities revealed that a DOB antigen-based IgG ELISA has to be used in sero-epidemiological studies; 7.1% (11/155) of the acute phase/early convalescent sera and 12.5% (2/16) of the late convalescent sera, respectively, reacted only with the homologous DOB antigen.

Isolation and characterization of Puumala hantavirus from Norway: evidence for a distinct phylogenetic sublineage.

Puumala (PUU) hantavirus is the aetiological agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome, which occurs in Fennoscandia, central Europe and Russia. In Norway, NE-like disease has been reported since 1946 and about 50 cases are diagnosed annually; however, the causative agent has not been characterized. In this study, a virus originating from bank voles (Clethrionomys glareolus) trapped near the town of Eidsvoll (Akershus county) was isolated and passaged in laboratory-bred bank voles. The bank vole strain was identified as a PUU virus by serological typing and by sequence analysis of the S and M gene segments. For comparison, complete or partial S sequences were determined for wild-type PUU strains from five locations in Sweden, two inhabited by the southern variant of bank vole present in Fennoscandia, and three by the northern variant. Phylogenetic analysis showed that Norwegian PUU strains are clustered together with Swedish strains from the first group forming a well-supported sublineage within the PUU genotype, distinct from other sublineages from northern Sweden, Finland, Russia and France. The results are consistent with the view of a complex evolutionary history of PUU strains in post-glacial Fennoscandia. Analyses of the current collection of nucleotide sequences suggest that PUU is the most variable genotype of the known hantaviruses.

Chimaeric HBV core particles carrying a defined segment of Puumala hantavirus nucleocapsid protein evoke protective immunity in an animal model.

Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carriers of foreign protective epitopes.

Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica).

Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment.

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.

Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family.

Characterization of Puumala virus nucleocapsid protein: identification of B-cell epitopes and domains involved in protective immunity.

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.