Tula Virus Research Articles

Prevalence of hantavirus infections in Germany

ZusammenfassungHantaviren gehören zur Gruppe der so genannten „emerging viruses“. Die in Europa vorkommenden humanpathogenen Viren rufen ein als „hämorrhagisches Fieber mit renalem Syndrom“ bezeichnetes Krankheitsbild unterschiedlicher Schweregrade hervor. Für die Diagnostik von Hantavirusinfektionen werden in der Regel Immunfluoreszenztests unter Verwendung Virus-infizierter Zellen oder Enzymimmunoassays und Immunoblots unter Verwendung rekombinanter Nukleokapsidproteinderivate eingesetzt. Für einen hochsensitiven Nachweis im Enzymimmunoassay ist der Einsatz des jeweiligen homologen Hantavirus-Nukleokapsidproteins notwendig. Zur serologischen Typisierung eignen sich Neutralisationstests, die jedoch in einigen Fällen nur bei Verwendung von Seren aus der späten Rekonvaleszenzphase aussagefähige Ergebnisse liefern. Die Seroprävalenz der Normalbevölkerung in Deutschland beträgt etwa 1%. Bei beruflich exponierten Risikogruppen, wie z. B. Waldarbeitern, wurde eine gegenüber der Normalbevölkerung höhere Seroprävalenz beobachtet. Endemiegebiete für Hantavirusinfektionen befinden sich insbesondere in Baden-Württemberg. In den Jahren 2001–2003 wurden bundesweit jährlich ca. 200 klinisch apparente Hantavirusinfektionen gemeldet. Ergebnisse von Neutralisationstests haben bisher nur Hinweise auf das Vorkommen von humanen Puumala- und Dobravavirusinfektionen, in sehr seltenen Fällen auch auf Infektionen mit Tulavirus gegeben. Bisher wurden in Deutschland nur milde bis moderate Verläufe von Hantavirusinfektionen beobachtet. Bis zu 10% der jährlich gemeldeten, klinisch apparenten Hantavirusinfektionen gehen auf importierte Infektionen zurück. Gegenwärtig liegen nur wenige molekulargenetische Befunde zur Verbreitung von Hantaviren in Deutschland vor. Weitere Untersuchungen an Patienten und bei Nagetieren sind notwendig, um diesbezüglich präzisere Informationen zu erlangen und die Gefährdung der Bevölkerung genauer beurteilen zu können.

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses.

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.

Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein.

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191-4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10(4) copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L-EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L-EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

Mapping of the regions involved in homotypic interactions of Tula hantavirus N protein.

Hantavirus nucleocapsid (N) protein has been suggested to form homodimers and homotrimers that are further integrated into the nucleocapsid filaments around the viral RNA. Here we report detailed mapping of the regions involved in the homotypic N protein interactions in Tula hantavirus (TULV). Peptide scan screening was used to define the interaction regions, and the mammalian two-hybrid assay was used for the functional analysis of N protein mutants. To study linear regions responsible for N protein interaction(s), we used peptide scanning in which N peptides synthesized on membranes recognize recombinant TULV N protein. The data showed that the N protein bound to membrane-bound peptides comprising amino acids 13 to 30 and 41 to 57 in the N-terminal part and 340 to 379, 391 to 407, and 410 to 419 in the C-terminal part of the molecule. Further mapping of the interaction regions by alanine scanning indicated the importance of basic amino acids along the N protein and especially asparagine-394, histidine-395, and phenyalanine-396 in forming the binding interface. Analysis of truncated mutants in the mammalian two-hybrid assay showed that N-terminal amino acids 1 to 43 are involved in and C-terminal amino acids 393 to 398 (VNHFHL) are absolutely crucial for the homotypic interactions. Furthermore, our data suggested a tail-to-tail and head-to-head binding scheme for the N proteins.

Non-covalent interaction between nucleocapsid protein of Tula hantavirus and small ubiquitin-related modifier-1, SUMO-1

To find cellular binding counterparts for the nucleocapsid protein (N) of Tula hantavirus (TULV), two cDNA libraries were screened using yeast two-hybrid systems based on LexA and Gal4 transcription factors. Five cDNA clones encoding SUMO-1 ( Small Ubiquitin-related MOdifier, also known as sentrin) were selected in the LexA system. Confocal microscopy revealed that, in infected cells, TULV N protein and SUMO-1 colocalize at the perinuclear area providing further evidence for interaction between the two proteins. Neither endogenous nor transiently expressed SUMO-1 was found to be covalently linked to the N protein. Additional evidence that the interaction is non-covalent was obtained in immunoprecipitation experiments: N protein-specific antibodies precipitated SUMO-1 from TULV-infected Vero E6 cell lysate. By using a pepscan assay, two basic amino acid stretches in the N-terminal part of SUMO-1 were shown to be involved in the interaction.

Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover.

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.

Tula hantavirus in Belgium.

European common voles (Microtus arvalis), captured in Belgium in 1999, were proven by molecular as well as by serological techniques to be infected with Tula hantavirus (TULV). This is the first evidence for the presence of TULV in this country. No indication of spill-over infections of Puumala virus, known to be highly endemic among bank voles (Clethrionomys glareolus) within the same geographical regions as the trapped TULV-infected common voles, was observed. Together with previous reports on the circulation of TULV in eastern/central Europe, this finding suggests a more wide-spread circulation of this hantavirus serotype throughout the continent.

Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment.

Since the discovery of RNA recombination in polioviruses, there has been a general belief that this mechanism operates only in positive-sense RNA viruses. Recently, studying wild-type Tula hantavirus, we observed a mosaic-like structure of the S RNA segment that was consistent with generation by recombination between viruses from two genetic lineages. Here we show transfection-mediated rescue of Tula virus carrying recombinant S RNA segment. Independent attempts yielded S RNA molecules of similar structure; the majority of them carried a break point located close to one of the break points suggested for natural recombinants. Recombinant virus purified from the original variant was able to grow to the same titers in cell culture and showed the same characteristic immunofluorescence pattern when stained for the nucleocapsid protein. While competent, the recombinant virus appeared to be slightly less competitive than the wild type. Sequence analysis of the S cDNA clones obtained from the purified recombinant virus confirmed that all S RNA molecules were of recombinant origin. This provides the first example of a negative-sense RNA virus constructed using homologous recombination.

Identification of Tula hantavirus in Pitymys subterraneus captured in the Cacak region of Serbia-Yugoslavia

Background: Atypical serum neutralizing antibody responses to prototype strains of Puumala viruses in some patients with hemorrhagic fever with renal syndrome (HFRS) have long suggested the existence of other hantaviruses in the Balkans. Objective: To determine the presence of arvicolid rodent-borne Puumala-like hantaviruses in Yugoslavia. Materials and Methods: Using reverse transcript-polymerase chain reaction, Tula virus RNA was amplified from lung tissues of a European pine vole ( Pitymys subterraneus) captured in 1987, following an outbreak of HFRS in the Cacak region of Serbia-Yugoslavia. Results: Sequence analysis of the entire coding region of the S segment and a 948-nucleotide region of the G2 glycoprotein-encoding M segment revealed divergence of approximately 14% from Tula virus strains harbored by European common voles ( Microtus arvalis) captured in Central Russia and the Czech Republic. However, nearly complete identity was found in the corresponding deduced amino acid sequences. Moreover, phylogenetic trees constructed by the maximum parsimony and neighbor-joining methods indicated that this Pitymys-borne hantavirus shared a common ancestry with other Tula virus strains. Conclusions: The data demonstrate that Pitymys subterraneus also serves as a rodent reservoir of Tula virus in Serbia-Yugoslavia. To what extent this represents virus spillover from Microtus arvalis warrants further investigation.

Genetic evidence for tula virus in Microtus arvalis and Microtus agrestis populations in Croatia.

To determine the threat of hantavirus infection to U.S. Forces, small mammals were sampled from training areas within Croatia. Of the 152 samples, 20 were positive for Tula virus (TUL), 12 common voles (Microtus arvalis) and eight field voles (Microtus agrestis). Sequences from M. agrestis were found in five and sequences from M. arvalis were found in six of seven sequence groups. The high percentage of the same TUL sequences in M. agrestis and M. arvalis suggests the co-occurrence of this virus in both Microtus species is not an accident. If M. agrestis field voles were accidentally infected with TUL, the percentage of polymerase chain reaction-positive animals should be lower than that of M. arvalis. Because the infection rate in M. arvalis (11.8%) was less than half of that found in M. agrestis (27.6%), it is unlikely that the predominance of positive M. agrestis could be due to accidental exposure. It is much more likely that the Croatian virus is circulating between both rodent species.

Short report: simultaneous occurrence of Dobrava, Puumala, and Tula Hantaviruses in Slovakia.

The prevalence of antibody to hantaviruses in Slovakia (serum panel n = 2,133) was lower in the western part (0.54%) and higher in the eastern part (1.91%) of the country and was found to be significantly enhanced in a group of forest workers from eastern Slovakia (5.88%). One-third of the IgM-negative convalescent phase sera from patients with hemorrhagic fever with renal syndrome exhibited antibodies reacting predominantly with Puumala virus antigen, while two-thirds had antibodies directed mainly against Hantaan virus antigen. Fine analysis of two Hantaan virus-reactive sera by a focus reduction neutralization test showed that Dobrava hantavirus was the source of these human infections. Initial results of rodent screening indicated the circulation of Dobrava virus in populations of striped field mice (Apodemus agrarius) in eastern Slovakia.

Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia.

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3'-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.

Completion of the Tula hantavirus genome sequence: properties of the L segment and heterogeneity found in the 3' termini of S and L genome RNAs.

In this study the L segment and the 5' and 3' termini of the S, M and L segments of the prototype Tula hantavirus (TUL) were sequenced, thus completing the first determination of the genome sequence of a hantavirus that has not been linked to any human disease. The TUL L segment comprises 6541 nt with one ORF of 6459 nt in the antigenome sense. This ORF potentially encodes a 2153 aa protein with a predicted molecular mass of 247 kDa. The amino acid sequence includes all the motifs conserved in RNA-dependent RNA polymerases. The 5' termini of all three genome RNAs (vRNAs) had the expected sequences conserved in hantaviruses. The 3' termini of M vRNAs were also conserved. However, the 3' termini of S and L vRNAs were heterogeneous as most of the sequenced 3' termini had either deletions of 1 to 22 nt or an extra 1 to 3 nt. No increase in the level of heterogeneity was seen in vRNAs of virions collected 3, 6, 9 and 12 days post-infection, suggesting that the heterogeneity already exists at the early stages of infection. The S and L vRNAs from infected cells had more truncated 3' termini than vRNAs from pelleted virus. Heterogeneity of the 3' termini of genome RNAs could decrease the efficiency of antigenome and mRNA syntheses and contribute to the slow growth observed for TUL and other hantaviruses in cell culture.

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

A panel of seven human monoclonal Fabs against Puumala virus (PUU) nucleocapsid protein (N) was obtained by panning an antibody phage-display library prepared from the spleen of a PUU-immune individual. Three antibodies reacted in immunoblotting and cross-reacted strongly with Tula and Sin Nombre virus recombinant N proteins. These antibodies mapped to the amino terminus of the N protein. One PUU glycoprotein 2 (G2)-specific Fab obtained against a novel epitope (G2c) cross-reacted with Khabarovsk virus but not with the other hantavirus serotypes. An N protein-specific Fab was successfully used as capture antibody to detect PUU-specific serum IgG and IgM antibodies in an enzyme immunoassay. The result demonstrates the usefulness of recombinant human Fabs as potential diagnostic tools.

Puumala virus and two genetic variants of Tula virus are present in Austrian rodents.

Puumala and Tula viruses are hantaviruses found in Europe and are associated with the rodents Clethrionomys glareolus and Microtus arvalis, respectively. Puumala virus is associated with the human disease nephropathia epidemica. In Austria, ten clinically diagnosed cases of nephropathia epidemica, presumably caused by Puumala virus infection, have been reported but not virologically confirmed [Leschinskaya et al., 1991; Aberle et al., 1996]. To identify the hantaviruses that are present in Austria, five species of rodents were trapped and screened for virus antibodies, antigen, and RNA. Hantaviruses were detected in two species, Cl. glareolus and M. arvalis, by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products from Cl. glareolus tissues yielded a unique Puumala virus sequence distinct from Puumala virus sequences reported from other parts of Europe. RT-PCR products from M. arvalis tissues yielded two genetically distinct Tula virus sequences, one similar to sequences reported from Slovakia and the Czech Republic and another that appears to be a novel genetic variant of Tula virus. This is the first confirmed report of hantaviruses in Austria.

IgE-based assay for early detection of HIV-1 infection in infants

5Serum samples were collected from patients with HFRS in the Tula region during the outbreak and from convalescent patients (collected between 1 month and 4 years after disease) in the Tula and Ryazan regions. Initial serological screening by immunofluorescence assay (IFA) revealed higher antibody titres to Hantaan, Seoul, 5 and DOB (N Apekina, unpublished) antigens than to Puumala. These findings indicated that a hantavirus other than Puumala virus was the causative agent of the outbreak. In our study of 22 hantavirus-IgG-positive serum samples, as confirmed by ELISA with Puumala, DOB, and Hantaan antigens, 2 all showed the highest reactivities to DOB. However, since previous studies have identified high levels of crossreactive antibodies to several hantaviruses by IFA or ELISA analysis, the serum samples were further assayed by focus-reduction neutralisation test which allows precise serotyping. To avoid misleading crossreactivity of neutralising antibodies during the acute phase of HFRS, 2 only samples from convalescent patients obtained more than 1 month after onset of disease, were selected. Nine such samples were analysed against the hantaviruses known to cause HFRS and the putatively apathogenic Tula virus (table). All serum samples showed high specificities of neutralising antibodies to DOB, confirming its involvement in the Tula-Ryazan outbreak. Our results do not confirm the previously proposed involvement of Seoul virus in the outbreak because none of the serum samples showed significant titres to this hantavirus.

Quasispecies in wild-type tula hantavirus populations.

Tula virus (TUL) is a recently detected hantavirus carried by European common voles. Reverse transcriptase PCR cloning was used to study TUL S segment/N protein quasispecies. Both the distribution and character of mutations observed in three mutant spectra indicated limited selection at the protein level. At least 8% of the mutations were neutral or well tolerated; fixation of such mutations may play a role in TUL evolution in its natural host.

Isolation and characterization of Tula virus, a distinct serotype in the genus Hantavirus, family Bunyaviridae.

A Vero E6 cell culture isolate of Tula virus (TUL), a hantavirus first detected in European common voles (Microtus arvalis and M. rossiaemeridionalis) by RT-PCR was obtained after initial passaging of TUL-infected vole lung samples in laboratory-colonized M. arvalis. TUL was defined as a classical serotype by a cross-focus-reduction neutralization test (FRNT) and was also shown to be distinct from other hantaviruses by haemagglutination inhibition assay. The sequences of S, M and partial L genome segments of the isolate were determined: the S segment was 99.9% identical to the original rodent-derived sequence. Serological evidence for a previous TUL infection was obtained from the serum of a blood donor living near a TUL focus in Moravia, Czech Republic, showing at least a 16-fold higher FRNT titre to TUL as compared to Puumala or other hantaviruses.

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 NH 2-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 NH 2-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.

Inhibition of Puumala and Tula Hantaviruses in Vero Cells by MxA Protein

Human MxA protein is a type I interferon-inducible intracytoplasmic protein, which mediates antiviral actions against a variety of negative-strand RNA viruses including influenza A, measles, and vesicular stomatitis viruses. Recently, it has also been shown that several members of theBunyaviridaefamily are inhibited by MxA protein. The hantavirus genus in theBunyaviridaefamily includes important human pathogenic viruses, e.g., Puumala (PUUV), Hantaan, and Sin Nombre viruses. Tula virus (TULV) is a new member of the genus, but its pathogenicity in man remains to be determined. As assumed by the similarities in replication strategy, MxA would be a good candidate molecule for antiviral action against these viruses, also. To gain more insight into the MxA action on PUUV, we studied PUUV and TULV replication in stably MxA gene-transfected Vero cells. We show that MxA protein has the capacity to inhibit both viral protein and RNA accumulation in virus-infected cells. We also studied PUUV and TULV infection in MxA-transfected U-937 cell clones. In these cell lines both hantaviruses grew poorly, independent of whether the cells were expressing MxA or not. Whether cell line-specific differences in the antiviral activity of MxA protein against hantaviruses exist cannot be conclusively determined due to the lack of productive infection of PUUV and TULV in U-937 cells.