Jan Hörling

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera

Hantavirus infection was diagnosed serologically by μ‐capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia‐Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute‐ or early convalescent‐phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute‐phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross‐neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala‐ and Dobrava‐like virus infections. RT‐PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow‐necked field mouse (Apodemus flavicollis). Cross‐FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala‐ and Dobrava‐like viruses caused HFRS in Bosnia‐Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found. J. Med. Virol. 53:51–59, 1997.

Characterization of tick-borne enchephalitis virus from Latvia

Viruses of the tick‐borne encephalitis (TBE) antigenic complex, within the family Flaviviridae, cause a variety of diseases including uncomplicated febrile illness, encephalitis, meningo‐encephalitis, hemorrhagic fever and chronic disease in humans, domesticated animals or wildlife species. TBE is a serious problem in Latvia with up to a 1,000 patients confirmed serologically annually 1994–1995. No previous data had been reported on the causative agent of TBE in Latvia. In the present study, a virus was isolated from serum of a patient with clinical symptoms of an acute TBE infection. Nucleotide sequence information obtained by direct reverse transcription‐polymerase chain reaction (RT‐PCR) and the serological characteristics of the isolated virus strain, designated TBE‐Latvia‐1‐96, indicated a closer relationship to the Vasilchenko strain, isolated in Novosibirsk (Siberia, Russia), as compared to the western European or far eastern subtypes of TBE viruses. In a mouse neurovirulence assay, a significant difference in survival rates (days) was shown between Latvia‐1‐96 and the western European TBE virus subtype. J. Med. Virol. 60:216–222, 2000.

The humoral response to Puumala virus infection (nephropathia epidemica) investigated by viral protein specific immunoassays

SummaryEnzyme-linked immunosorbent assays for determination of antibodies directed to the nucleocapsid protein (N) or to either of the two envelope glycoproteins (G1 and G2) of Puumala virus were designed and evaluated. The assays were proven to be entirely restricted for each viral structural protein by biotin-labelled monoclonal antibodies. Sera from sequentially bled nephropathia epidemica patients (acute, convalescent, and 2-year sera) and sera from 10–20 year convalescents were examined for antibody specificity. All but one (n=19) acute phase sera were shown to contain IgM antibodies directed to all three viral proteins. In the convalescent specimens the proportions of IgM to the different viral components were similar, but lower, when compared to the acute samples. Low levels of IgM against N and G2 were found in two out of ten 2-year sera. No virus-specific IgM were detected in sera drawn 10–20 years after infection. IgG antibodies to all three viral proteins were detected in all except one acute phase serum. The IgG response initially increased more rapidly to N, as compared to the anti-glycoprotein responses. The levels of glycoprotein-specific IgG were considerably increased 2 years after the disease, when compared to the levels detected in the convalescent specimens. The levels and specificities of IgG in very late convalescent sera (drawn 10–20 years after disease) resembled those detected 2 years after infection.

Khabarovsk virus: a phylogenetically and serologically distinct Hantavirus isolated from Microtus fortis trapped in far-east Russia

Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR).

Distribution and genetic heterogeneity of Puumala virus in Sweden

Small mammals trapped in Sweden were analysed for specific antibody responses against three hantavirus serotypes and for the presence of viral antigen. To determine the genetic identity of viral RNA in lungs of seropositive bank voles (Clethrionomys glareolus), polymerase chain reactions and subsequent partial sequencing of both the M and S segments were employed. The sequences obtained were all identified as Puumala (PUU) virus, with a high degree of heterogeneity between the different geographical localities. Alignment of nucleotide and deduced amino acid sequences, together with phylogenetic analysis, showed that PUU viruses circulating in central Sweden were distinct from those in the northern region. The localization of the two distinct PUU virus genotypes was shown to correlate with the postglacial recolonization of Sweden by bank voles.

Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidemica: a novel method using antigen-coated magnetic beads for specific B cell isolation.

Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein-Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four anti-glycoprotein 2-specific clones. Identical isotypes (IgGl lambda) and isoelectric points (9.2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partner SPAM-8. Several of the established human x (human x mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.

Antibodies to Puumala virus in humans determined by neutralization test

An assay for detection of neutralizing antibodies to Puumala virus using 96-well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 1-4 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected 10-20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NT-ELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests.

Single amino acid substitutions in Puumala virus envelope glycoproteins G1 and G2 eliminate important neutralization epitopes

Two monoclonal antibody escape virus mutants (MARs), rescued from a human MAb to glycoprotein 2 (G2) and a bank vole monoclonal antibody (MAb) directed to glycoprotein 1 (G1) of Puumala virus, strain Sotkamo, were produced by using a combination of neutralization tests and antigen detection. The MARs and the original virus were analyzed by nucleotide sequencing and the responsible mutations were defined and characterized. The G1 mutation was found to constitute an A to T nucleotide substitution, giving raise to an aspartic acid to valine mutation at residue 272, potentially increasing the hydrophobicity of this region. The G2 mutation was found to constitute a C to T substitution, altering the residue 944 from serine into the more hydrophobic phenylalanine and resulting in secondary structure alterations. The mutation was found to be in close vicinity to a glycosylation site. Synthetic peptides covering the regions of the native virus, defined by the MARs, were produced and evaluated for reactivity with the corresponding MAb. The peptides were not recognized by the MAbs, and did not inhibit the binding of the MAbs in competition assays. Sera from mice immunized with the peptides were not able to recognize the native protein. This indicates that the epitopes are non-linear and/or glycosylated in the native state, or alternatively, that the G1 and G2 MAbs binds to regions away from the mutations.

Nucleotide and deduced amino acid sequences of the M and S genome segments of a Swedish Puumala virus isolate

The Swedish Puumala (PUU) virus strain Vindeln 83-L20, isolated from a bank vole trapped in 1983 near Vindeln, Västerbotten county, Sweden, was characterized by nucleotide sequence analysis. The coding region of the M segment was determined by PCR followed by direct sequencing and the entire S segment was characterized by cloning and nucleotide sequence analysis. The genomic organization was found to be very similar to that of other PUU virus strains regarding open reading frames, polypeptide sizes and potential glycosylation sites. According to phylogenetic analysis 83-L20 was found to represent a new lineage within the Puumala virus serotype in the Hantavirus genus. The M segment sequence of 83-L20 was found to be more closely related to the Finnish PUU virus strains than to strains from Central Europe or from Russia. The evolutionary origin of the S segment was not as clearly resolved since the branching points of all PUU virus strains in the phylogenetic tree were nearly the same.