Tula Virus Research Articles

254 Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter

254 Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter

Hantavirus Outbreak, Germany, 2007

Hantavirus Outbreak, Germany, 2007

New Genetic Lineage of Tula Hantavirus in Microtus arvalis obscurus in Eastern Kazakhstan

Genomic sequences of Tula (TULV) hantavirus were recovered from tissue samples of European common voles Microtus arvalis (subspecies obscurus) captured in Kazakhstan, Central Asia. Phylogenetic analysis of the S genomic segment of Kazakh TULV strains showed that they form distinct, well supported genetic lineage and share a more ancient common ancestor with two Russian lineages of TULV. The deduced sequence of the nucleocapsid (N) protein of Kazakh TULV strains carried specific amino acid signature: T274Q276T281. The Microtus arvalis group includes several sibling species and/or subspecies in Eurasia, indicating recent and ongoing evolutionary radiation. Our data on TULV lineages in Central Asia, the region not studied for hantaviruses earlier, highlight the diversity of both Microtus host and the virus and also their co-evolution.

High Rates of Molecular Evolution in Hantaviruses

Hantaviruses are rodent-borne Bunyaviruses that infect the Arvicolinae, Murinae, and Sigmodontinae subfamilies of Muridae. The rate of molecular evolution in the hantaviruses has been previously estimated at approximately 10(-7) nucleotide substitutions per site, per year (substitutions/site/year), based on the assumption of codivergence and hence shared divergence times with their rodent hosts. If substantiated, this would make the hantaviruses among the slowest evolving of all RNA viruses. However, as hantaviruses replicate with an RNA-dependent RNA polymerase, with error rates in the region of one mutation per genome replication, this low rate of nucleotide substitution is anomalous. Here, we use a Bayesian coalescent approach to estimate the rate of nucleotide substitution from serially sampled gene sequence data for hantaviruses known to infect each of the 3 rodent subfamilies: Araraquara virus (Sigmodontinae), Dobrava virus (Murinae), Puumala virus (Arvicolinae), and Tula virus (Arvicolinae). Our results reveal that hantaviruses exhibit short-term substitution rates of 10(-2) to 10(-4) substitutions/site/year and so are within the range exhibited by other RNA viruses. The disparity between this substitution rate and that estimated assuming rodent-hantavirus codivergence suggests that the codivergence hypothesis may need to be reevaluated.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF

BackgroundThe competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells.ResultsIn IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66–67 aa).ConclusionOur data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

Hantaviruses and TNF-alpha act synergistically to induce ERK1/2 inactivation in Vero E6 cells

BackgroundWe have previously reported that the apathogenic Tula hantavirus induces apoptosis in Vero E6 epithelial cells. To assess the molecular mechanisms behind the induced apoptosis we studied the effects of hantavirus infection on cellular signaling pathways which promote cell survival. We previously also observed that the Tula virus-induced cell death process is augmented by external TNF-α. Since TNF-α is involved in the pathogenesis of hantavirus-caused hemorrhagic fever with renal syndrome (HFRS) we investigated its effects on HFRS-causing hantavirus-infected cells.ResultsWe studied both apathogenic (Tula and Topografov) and pathogenic (Puumala and Seoul) hantaviruses for their ability to regulate cellular signaling pathways and observed a direct virus-mediated down-regulation of external signal-regulated kinases 1 and 2 (ERK1/2) survival pathway activity, which was dramatically enhanced by TNF-α. The fold of ERK1/2 inhibition correlated with viral replication efficiencies, which varied drastically between the hantaviruses studied.ConclusionWe demonstrate that in the presence of a cytokine TNF-α, which is increased in HFRS patients, hantaviruses are capable of inactivating proteins that promote cell survival (ERK1/2). These results imply that hantavirus-infected epithelial cell barrier functions might be compromised in diseased individuals and could at least partially explain the mechanisms of renal dysfunction and the resulting proteinuria seen in HFRS patients.

Kinship, dispersal and hantavirus transmission in bank and common voles

Hantaviruses are among the main emerging infectious agents in Europe. Their mode of transmission in natura is still not well known. In particular, social features and behaviours could be crucial for understanding the persistence and the spread of hantaviruses in rodent populations. Here, we investigated the importance of kinclustering and dispersal in hantavirus transmission by combining a fine-scale spatiotemporal survey (4 km2) and a population genetics approach. Two specific host-hantavirus systems were identified and monitored: the bank vole Myodes, earlier Clethrionomys glareolus--Puumala virus and the common vole Microtus arvalis--Tula virus. Sex, age and landscape characteristics significantly influenced the spatial distribution of infections in voles. The absence of temporal stability in the spatial distributions of viruses suggested that dispersal is likely to play a role in virus propagation. Analysing vole kinship from microsatellite markers, we found that infected voles were more closely related to each other than non-infected ones. Winter kin-clustering, shared colonies within matrilineages or delayed dispersal could explain this pattern. These two last results hold, whatever the host-hantavirus system considered. This supports the roles of relatedness and dispersal as general features for hantavirus transmission.

Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon‐beta promoter

The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs.

Puumala and Tula hantaviruses in France

The first genome sequences of Tula (TULV) and Puumala (PUUV) hantaviruses undoubtedly originated from France were recovered from tissue samples of European common voles and bank voles captured in Jura region. Genetic analysis of S and M segments of French PUUV strain revealed its highest similarity to strains from neighboring Belgium and Germany and also from Slovakia. On phylogenetic trees, French PUUV strain was placed within the central European lineage formed by strains from these three countries. Both of our French TULV strains clustered together and formed a distinct, well-supported genetic lineage.

Chemokine Production Predominates in Human Monocytes Infected with Tula Virus

Many reports suggest the hypothesis of a complex immune response accompanying hantaviral infections. However, little is known about the immunopathogenesis of nonpathogenic hantaviruses, especially Tula virus (TULV). The aim of our study was to determine the cytokine/chemokine profile induced after the infection of human macrophages with TULV and the role of viral replication in this process. Also, we wanted to establish how the study of TULV is relevant to our previous study of pathogenic hantaviruses. We showed that TULV-infected macrophages produced chemokines (interleukin-8, macrophage chemoattractant protein-1, and macrophage inflammatory protein-1beta) important for recruiting inflammatory cells, whereas no significant changes were recorded in the tested cytokine levels. This property was not influenced by ultraviolet inactivation. There were some differences in chemokine production compared with our previous study with pathogenic hantaviruses. A possible explanation could be a different way of entering host cells found in the pathogenic and nonpathogenic hantaviruses and activation of different intracellular signaling pathways.

Novel Hantavirus Sequences in Shrew, Guinea

Novel Hantavirus Sequences in Shrew, Guinea

Regions of importance for interaction of puumala virus nucleocapsid subunits

Puumala virus (PUUV) is a hantavirus that causes a mild form of hemorrhagic fever with renal syndrome in northern and central Europe, and in large parts of Russia. The nucleocapsid (N) protein encoded by hantaviruses plays an important role in the life-cycle of these viruses, and one important function for the N-protein is to oligomerize, surround and protect the viral RNAs. We have identified amino- and carboxy-terminal regions involved in PUUV N-N interactions, which comprise amino acids 100-120 and 330-405. Our findings strengthen the hypothesis that the amino-terminus of the N-protein of hantaviruses holds a more regulatory function regarding N-N interactions, while conserved residues in the carboxy-terminal region, F335 together with F336 and W392, in concert with Y388 and/or F400 seems to play a more critical role in the PUUV N-N formation. This study provides evidence that the amino-terminal regions involved in the N-N interaction of Puumala virus are similar to those reported for Seoul virus (SEOV) and to some extent Hantaan virus (HTNV), even though the identity between PUUV N and SEOV/HTNV N is markedly lower than between PUUV N and Tula virus (TULV) N or Sin Nombre virus (SNV) N.

Oligomerization of Hantavirus Nucleocapsid Protein: Analysis of the N-Terminal Coiled-Coil Domain

Hantaviruses constitute a genus in the family Bunyaviridae. They are enveloped negative-strand RNA viruses with a tripartite genome encoding the nucleocapsid (N) protein, the two surface glycoproteins Gn and Gc, and an RNA-dependent RNA polymerase. The N protein is the most abundant component of the virion; it encapsidates genomic RNA segments forming ribonucleoproteins and participates in genome transcription and replication as well as virus assembly. In the course of RNA encapsidation, N protein forms intermediate trimers via head-to-head and tail-to-tail interactions. We analyzed the amino-terminal trimerization domain (amino acid residues 1 to 77) of Tula hantavirus using computer modeling, mammalian two-hybrid assay, and immunofluorescence assay. The results obtained were consistent with the existence of an antiparallel coiled-coil stabilized by interactions between hydrophobic residues. Residues L44, V51, and L58 were important for the N-N interaction; other residues, e.g., L25 and V32, also made a contribution, albeit a modest one. Our alignments of the N-terminal domain of the hantaviral N proteins suggest the coiled-coil structure, and hence the mode of N-protein oligomerization, is conserved among hantaviruses.

Prolonged survival of Puumala hantavirus outside the host: evidence for indirect transmission via the environment

The capability of rodent-borne viruses to survive outside the host is critical for the transmission dynamics within rodent populations and to humans. The transmission of Puumala virus (PUUV) in colonized bank voles (Clethrionomys glareolus) was investigated and additional longevity studies in cell culture with PUUV and Tula (TULV) hantaviruses were performed. Wild-type PUUV excreted by experimentally infected donor bank voles was shown to be transmitted indirectly between rodents through contaminated beddings, and maintained its infectivity to recipient voles at room temperature for 12-15 days. In cell culture supernatants, PUUV and TULV remained infectious for 5-11 days at room temperature and up to 18 days at 4 degrees C, but were inactivated after 24 h at 37 degrees C. Interestingly, a fraction of dried virus was still infectious after 1 h at 56 degrees C. These results demonstrated that hantavirus transmission does not require direct contact between rodents, or between rodents and humans, and that the indirect transmission of PUUV through contaminated environment takes place among the rodents for a prolonged period of time. The results also have implications for safety recommendations for work with hantaviruses and for preventive measures.

A novel method for cloning of non-cytolytic viruses

Hantaviruses are rodent-borne pathogens with a segmented single-stranded RNA genome of negative polarity. Spontaneous occurrence of variants with genetic heterogeneity have been observed both in vivo and in vitro. The objective of this study was to establish a method for the cloning of genetically homogenous hantaviruses which can be used for subsequent functional studies. Infected VeroE6 cells were incubated with an agarose/medium overlay to prevent uncontrolled distribution of de novo synthesized virus. Thereafter, the overlay was removed and stored for isolation of the diffused virus. The cell layer was fixed and viral antigen-containing foci were detected by immunochemistry. The relative location of the foci on the culture dish was used to trap individual virus clones in the corresponding overlay. The clones were picked and used for re-infection. According to this novel protocol three different hantaviruses, i.e. Hantaan, Puumala, and Tula virus, were purified. In the course of purification the titers of the resulting virus stocks were increased by 10–1000-fold. In addition, this method was used to purify a minor Puumala virus variant from a parental stock containing a mixture of two variants. Taken together, the method presented is well suited to isolate genetically homogenous hantaviruses and might also be applicable for other non-cytolytic viruses.

Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture

Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.

Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells

Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER–Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261–3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH 2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space

The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.

Characterization of Tula virus from common voles (microtus arvalis) in Poland: evidence for geographic-specific phylogenetic clustering.

Tula virus (TULV), a recently identified arvicolid rodent-borne hantavirus, is harbored by the European common vole (Microtus arvalis) in Central Russia and the Czech and Slovak Republics. We report the isolation and characterization of this hantavirus from M. arvalis captured in Poland, a country where human disease caused by hantaviruses has not been recognized. Of 34 arvicolid rodents (24 Clethrionomys glareolus, 9 M. arvalis, 1 Pitymys sp.) captured in Lodz and Tuszyn, Poland, during June to September 1995, sera from 3 M. arvalis and 3 C. glareolus contained IgG antibodies to Puumala virus (PUUV), as determined by an indirect immunofluorescent antibody assay. Alignment and comparison of the 1852-nucleotide S segment and a 1676-nucleotide region of the G2 glycoprotein-encoding M segment, amplified from lung tissues of two hantavirus-seropositive M. arvalis, revealed 83.9-85.2% and 82.3-83.5% sequence similarity, respectively, with TULV strains from Central Russia and the Czech and Slovak Republics. A > 98% sequence conservation was found at the amino acid level. Phylogenetic analysis indicated that the newly found TULV strains from Poland were closely related to, but distinct from, TULV from elsewhere in Europe.