Tula Virus Research Articles

Revised time scales of RNA virus evolution based on spatial information.

The time scales of pathogen evolution are of major concern in the context of public and veterinary health, epidemiology and evolutionary biology. Dating the emergence of a pathogen often relies on estimates of evolutionary rates derived from nucleotide sequence data. For many viruses, this has yielded estimates of evolutionary origins only a few hundred years in the past. Here we demonstrate through the incorporation of geographical information from virus sampling that evolutionary age estimates of two European hantaviruses are severely underestimated because of pervasive mutational saturation of nucleotide sequences. We detected very strong relationships between spatial distance and genetic divergence for both Puumala and Tula hantavirus-irrespective of whether nucleotide or derived amino acid sequences were analysed. Extrapolations from these relationships dated the emergence of these viruses most conservatively to at least 3700 and 2500 years ago, respectively. Our minimum estimates for the age of these hantaviruses are ten to a hundred times older than results from current non-spatial methods, and in much better accordance with the biogeography of these viruses and their respective hosts. Spatial information can thus provide valuable insights on the deeper time scales of pathogen evolution and improve our understanding of disease emergence.

High Prevalence of Tula Hantavirus in Common Voles in The Netherlands.

Tula virus (TULV) is a zoonotic hantavirus. Knowledge about TULV in the Netherlands is very scarce. Therefore in 2014, 49 common voles (Microtus arvalis) from a region in the south of the Netherlands, and in 2015, 241 common voles from regions in the north of the Netherlands were tested with the TULV quantitative RT-PCR. In the southern region, prevalence of TULV was 41% (20/49). In the northern regions, prevalence ranged from 12% (4/34) to 45% (17/38). Phylogenetic analysis of the obtained sequences showed that the regions fall within different clusters. Voles from the south were also tested on-site for the presence of hantavirus antibodies, but serology results were poorly associated with qRT-PCR results. These findings suggest that TULV may be more widespread than previously thought. No human TULV cases have been reported thus far in the Netherlands, but differentiation between infection by TULV or the closely related Puumala virus is not made in humans in the Netherlands, thus cases may be misdiagnosed.

Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis).

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1-L3, M2-M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

A Molecular-Level Account of the Antigenic Hantaviral Surface.

SummaryHantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential

Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level.

High genetic structuring of Tula hantavirus.

Tula virus (TULV) is a vole-associated hantavirus with low or no pathogenicity to humans. In the present study, 686 common voles (Microtus arvalis), 249 field voles (Microtus agrestis) and 30 water voles (Arvicola spec.) were collected at 79 sites in Germany, Luxembourg and France and screened by RT-PCR and TULV-IgG ELISA. TULV-specific RNA and/or antibodies were detected at 43 of the sites, demonstrating a geographically widespread distribution of the virus in the studied area. The TULV prevalence in common voles (16.7 %) was higher than that in field voles (9.2 %) and water voles (10.0 %). Time series data at ten trapping sites showed evidence of a lasting presence of TULV RNA within common vole populations for up to 34 months, although usually at low prevalence. Phylogenetic analysis demonstrated a strong genetic structuring of TULV sequences according to geography and independent of the rodent species, confirming the common vole as the preferential host, with spillover infections to co-occurring field and water voles. TULV phylogenetic clades showed a general association with evolutionary lineages in the common vole as assessed by mitochondrial DNA sequences on a large geographical scale, but with local-scale discrepancies in the contact areas.

Tula hantavirus in Crimea

Genetic evidence of the Tula virus (TULV) in Crimea region of Russia is presented. Based on the reverse transcription PCR and subsequent sequence analysis, a total of 4 RNA isolates of the TULV were identified from the tissue samples of the Altai voles Microtus obscurus captured in the Bakhchisaray district of the Republic Crimea. Phylogenetic analysis of the S-, M-, and L-segment sequences of the Crimean TULV strains showed that they formed distinct genetic lineage, Russia IV, in the TULV variant. New sequences were most closely related to the lineage Russia I sequences obtained from common vole (M. arvalis) captured in the Tula region in Central Russia

Adler hantavirus, a new genetic variant of Tula virus identified in Major’s pine voles (Microtus majori) sampled in southern European Russia

Although at least 30 novel hantaviruses have been recently discovered in novel hosts such as shrews, moles and even bats, hantaviruses (family Bunyaviridae, genus Hantavirus) are primarily known as rodent-borne human pathogens. Here we report on identification of a novel hantavirus variant associated with a rodent host, Major’s pine vole (Microtus majori).Altogether 36 hantavirus PCR-positive Major’s pine voles were identified in the Krasnodar region of southern European Russia within the years 2008–2011. Initial partial L-segment sequence analysis revealed novel hantavirus sequences. Moreover, we found a single common vole (Microtusarvalis) infected with Tula virus (TULV). Complete S- and M-segment coding sequences were determined from 11 Major’s pine voles originating from 8 trapping sites and subjected to phylogenetic analyses.The data obtained show that Major’s pine vole is a newly recognized hantavirus reservoir host. The newfound virus, provisionally called Adler hantavirus (ADLV), is closely related to TULV. Based on amino acid differences to TULV (5.6–8.2% for nucleocapsid protein, 9.4–9.5% for glycoprotein precursor) we propose to consider ADLV as a genotype of TULV. Occurrence of ADLV and TULV in the same region suggests that ADLV is not only a geographical variant of TULV but a host-specific genotype. High intra-cluster nucleotide sequence variability (up to 18%) and geographic clustering indicate long-term presence of the virus in this region.

Multiple infections of rodents with zoonotic pathogens in Austria.

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the host's immune system, the impact of the host-pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

Surveillance of Hantaviruses in Poland: A Study of Animal Reservoirs and Human Hantavirus Disease in Subcarpathia

The first cluster of hemorrhagic fever with renal syndrome (HFRS) in Poland was identified in 2007 in the Subcarpathian region. The natural environment of this area is a key habitat for hantavirus vectors. The animal reservoir of existing human HFRS clusters was studied to assess the occurrence of viruses (including Tula virus, Puumala virus, and Dobrava-Belgrade virus) among rodents. We examined 70 suspected human cases with symptoms corresponding to the clinical picture of HFRS. Serological analysis (indirect immunofluorescence assay and immunoblot) confirmed the presence of anti-hantavirus antibodies in 18 patients, which were surveyed with regard to developed symptoms and presumed rodent contact. Seroepidemiological analysis of newly confirmed human cases was performed, putative areas of human exposure were studied, and 194 rodents were subsequently captured from identified areas. Internal organs (lungs, heart, spleen, bladder, and kidneys) were collected from 64 Apodemus flavicollis, 55 Apodemus agrarius, 40 Myodes glareolus, 21 Mus musculus, and 14 Microtus arvalis and tested for the presence of hantavirus RNA by reverse transcription and subsequent real-time PCR. Positive samples were also tested by indirect immunofluorescence. Animal reservoir surveillance enabled the first detection of Puumala virus and Dobrava-Belgrade virus among animals in Poland. Furthermore, some places where rodents were captured correlated with areas of residence of laboratory-confirmed human cases and likely detected virus species. Moreover, three species of hantaviruses coexisting in a relatively small area were identified.

Genetic detection of Dobrava-Belgrade hantavirus in the edible dormouse (Glis glis) in central Serbia.

Hantaviruses are endemic in the Balkans, particularly in Serbia, where sporadic cases and/or outbreaks of hantaviral human disease have been reported repeatedly, and evidenced serologically. Here, we present genetic detection of Dobrava-Belgrade virus (DOBV) hantaviral sequences in wild rodents trapped in central Serbia. All the animals were pre-screened serologically by indirect immunofluorescence (IF) test and only those with a positive finding of hantaviral antigens were further tested by polymerase chain reaction. Of the total of 104 trapped animals, 20 were found to be IF positive and of those three were positive for hantaviral RNA: one Microtus arvalis for Tula virus, and one each of Apodemus agrarius and Glis glis for DOBV. Phylogenetic analysis of the obtained sequences implies putative DOBV spillover infection of A. agrarius and G. glis from Apodemus flavicollis. However, future investigations should help to identify the most common natural host and geographical distribution of DOBV in its reservoir hosts in Serbia.

Phylogeographic Diversity of Pathogenic and Non-Pathogenic Hantaviruses in Slovenia

Slovenia is a very diverse country from a natural geography point of view, with many different habitats within a relatively small area, in addition to major geological and climatic differences. It is therefore not surprising that several small mammal species have been confirmed to harbour hantaviruses: A. flavicollis (Dobrava virus), A. agrarius (Dobrava virus–Kurkino), M. glareolus (Puumala virus), S. areanus (Seewis virus), M. agrestis, M. arvalis and M. subterraneus (Tula virus). Three of the viruses, namely the Dobrava, Dobrava–Kurkino and Puumala viruses, cause disease in humans, with significant differences in the severity of symptoms. Due to changes in haemorrhagic fever with renal syndrome cases (HFRS) epidemiology, a detailed study on phylogenetic diversity and molecular epidemiology of pathogenic and non-pathogenic hantaviruses circulating in ecologically diverse endemic regions was performed. The study presents one of the largest collections of hantavirus L, M and S sequences obtained from hosts and patients within a single country. Several genetic lineages were determined for each hantavirus species, with higher diversity among non-pathogenic compared to pathogenic viruses. For pathogenic hantaviruses, a significant geographic clustering of human- and rodent-derived sequences was confirmed. Several geographic and ecological factors were recognized as influencing and limiting the formation of endemic areas.

Hantaviruses in Germany: threat for zoo, pet, companion and farm animals?

Hantaviruses are so-called "emerging" and "re-emerging" viruses because of the new and sudden nature of their appearance. Human infections can lead to two distinct disease patterns, the Haemorrhagic Fever with Renal Syndrome and the Hantavirus Cardiopulmonary Syndrome. All known human pathogenic hantaviruses are transmitted through rodent hosts. There are three rodent-associated hantaviruses in Germany. The bank vole-associated Puumala virus (PUUV) is responsible for most of the human hantavirus infections. The Dobrava-Belgrade virus (DOBV) associated with the striped field mouse is causing hantavirus disease in the North and Northeast of Germany. The human pathogenicity of Tula virus (TULV) is still controversially discussed--the virus has been mainly associated with the common vole as the reservoir, but was molecularly detected also in the field and the water vole. More recently, two shrew-borne hantaviruses were described in Germany, i. e. Seewis virus in the common shrew and Asikkala virus in the pygmy shrew. Systematic studies about hantavirus infections of zoo, pet, companion and farm animals are still lacking. Hence, the aim of this review article is to summarise the current knowledge on this topic and raise the attention of veterinarians to potentially overlooked clinical disease patterns.

Tula Hantavirus Infection in Immunocompromised Host, Czech Republic

We report molecular evidence of Tula hantavirus as an etiologic agent of pulmonary-renal syndrome in an immunocompromised patient. Acute hantavirus infection was confirmed by using serologic and molecular methods. Sequencing revealed Tula virus genome RNA in the patient’s blood. This case shows that Tula virus can cause serious disease in humans.

Hypoxia Induces Permeability and Giant Cell Responses of Andes Virus-Infected Pulmonary Endothelial Cells by Activating the mTOR-S6K Signaling Pathway

Andes virus (ANDV) is a South American hantavirus that causes a highly lethal hantavirus pulmonary syndrome (HPS) characterized by hypoxia, thrombocytopenia, and vascular leakage leading to acute pulmonary edema. ANDV infects human pulmonary microvascular and lymphatic endothelial cells (MECs and LECs, respectively) and nonlytically enhances the permeability of interendothelial cell adherence junctions in response to vascular endothelial growth factor (VEGF). Recent findings also indicate that ANDV causes the formation of giant endothelial cells. Here, we demonstrate that hypoxic conditions alone enhance permeability and giant cell responses of ANDV-infected MECs and LECs through activation of the mTOR signaling pathway. In contrast to infection of cells with nonpathogenic Tula virus (TULV), we observed that exposure of ANDV-infected MECs and LECs to hypoxic conditions resulted in a 3- to 6-fold increase in monolayer permeability and the formation of giant cells 3× to 5× normal size. ANDV infection in combination with hypoxic conditions resulted in the enhancement of hypoxia-inducible factor 1α (HIF1α)-directed VEGF A, angiopoietin 4, and EGLN3 transcriptional responses. Constitutive mTOR signaling induces the formation of giant cells via phosphorylation of S6K, and mTOR regulates hypoxia and VEGF A-induced cellular responses. We found that S6K was hyperphosphorylated in ANDV-infected, hypoxia-treated MECs and LECs and that rapamycin treatment for 1 h inhibited mTOR signaling responses and blocked permeability and giant cell formation in ANDV-infected monolayers. These findings indicate that ANDV infection and hypoxic conditions enhance mTOR signaling responses, resulting in enhanced endothelial cell permeability and suggest a role for rapamycin in therapeutically stabilizing the endothelium of microvascular and lymphatic vessels during ANDV infection.

Evidence of recombination in Tula virus strains from Serbia

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

Novel Hantavirus in Wildlife, United Kingdom

Novel Hantavirus in Wildlife, United Kingdom

Taxonomie des hantavirus et situation des hantaviroses en France

Hantavirus taxonomy and situation in France Hantaviruses are viruses belonging to the family Bunyaviridae. Twenty-three species are officially reported but more than 70 taxa are not yet classified. Each virus taxa is usually associated with one natural host species, including rodents and soricomorphs. Sequences of new hantaviruses have also been recently detected in bats. Transmission of the virus between individuals occurred through direct contact or through inhalation of excretions or secretions. Using these routes, some rodents-borne hantaviruses can be transmitted to humans and cause a hemorrhagic fever with renal syndrome (HFRS) or a cardiopulmonary syndrome (HPS). There is no specific treatment of the diseases. Three of these zoonotic viruses have been detected in Metropolitan France. Puumala virus, hosted by Myodes glareolus, is medically the most important. About 1000 HFRS have been detected for the last ten years in the North-East part of France. Outbreaks occur in this endemic area every two or three years. A peak in the detection of cases is observed by the end of spring. Adults, mainly men, are concerned. Wood handling, opening and cleaning of unused building, forest activities are occupations favoring the exposure to the virus. Seoul virus has been detected in its reservoir Rattus norvegicus in Lyon. Human cases have been serologically suspected but the first virological evidence of a human case occurred only in 2012. Tula virus has been detected in its natural host Microtus arvalis in Jura. No human case has been detected. Lastly Maripa virus has been described for the first time in French Guiana. This virus has been responsible for three HPS cases, including two fatal cases. Thirty years after the first detection of PUUV human cases, a lot remains to discover in the circulation of these viruses in the country.

Tula Virus Infections in the Eurasian Water Vole in Central Europe

Recent reports of novel hantaviruses in shrews and moles and the detection of rodent-borne hantaviruses in different rodent species raise important questions about their host range and specificity, evolution, and host adaptation. Tula virus (TULV), a European hantavirus, is believed to be slightly or non-pathogenic in humans and was initially detected in the common vole Microtus arvalis, the East European vole M. levis (formerly rossiaemeridionalis), and subsequently in other Microtus species. Here we report the first multiple RT-PCR detection and sequence analyses of TULV in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. Additional novel TULV S-, M-, and L-segment sequences were obtained from M. arvalis and M. agrestis trapped in Germany at sites close to trapping sites of TULV-RT-PCR-positive water voles. Serological investigations using a recombinant TULV nucleocapsid protein revealed the presence of TULV-reactive antibodies in RT-PCR-positive and a few RT-PCR-negative water voles. Phylogenetic analyses revealed a geographical clustering of the novel S-, M-, and L-segment sequences from A. amphibius with those of M. arvalis- and M. agrestis-derived TULV lineages, and may suggest multiple TULV spillover or a potential host switch to A. amphibius. Future longitudinal studies of sympatric Microtus and Arvicola populations and experimental infection studies have to prove the potential of A. amphibius as an additional TULV reservoir host.

The cytoplasmic tail of hantavirus Gn glycoprotein interacts with RNA

We recently characterized the interaction between the intraviral domains of envelope glycoproteins (Gn and Gc) and ribonucleoprotein (RNP) of Puumala and Tula hantaviruses (genus Hantavirus, family Bunyaviridae). Herein we report a direct interaction between spike-forming glycoprotein and nucleic acid. We show that the envelope glycoprotein Gn of hantaviruses binds genomic RNA through its cytoplasmic tail (CT). The nucleic acid binding of Gn-CT is unspecific, as demonstrated by interactions with unrelated RNA and with single-stranded DNA. Peptide scan and protein deletions of Gn-CT mapped the nucleic acid binding to regions that overlap with the previously characterized N protein binding sites and demonstrated the carboxyl-terminal part of Gn-CT to be the most potent nucleic acid-binding site. We conclude that recognition of the RNP complex by the Gn-CT could be mediated by interactions with both genomic RNA and the N protein. This would provide the required selectivity for the genome packaging of hantaviruses.