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Viruses in the genus Orthohantavirus within the family Hantaviridae cause human hantavirus infections and represent a threat to public health. Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense (Puumala virus; PUUV), was first identified in Tobetsu, Hokkaido, Japan. Although it is genetically related to the prototype of PUUV, the evolutionary pathway of HOKV is unclear. We conducted a field survey in a forest in Tobetsu in 2022 and captured 44 rodents. Complete coding genome sequences of HOKVs were obtained from five viral-RNA-positive rodents (four Myodes rufocanus bedfordiae and one Apodemus speciosus). Phylogenetic analysis revealed a close relationship between the phylogenies and geographical origins of M. rufocanus-related orthohantaviruses. Comparison of the phylogenetic trees of the S segments of orthohantaviruses and the cytochrome b genes of Myodes species suggested that Myodes-related orthohantaviruses evolved in Myodes rodent species as a result of genetic isolation and host switching., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5-40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced. We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein. Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The objective is to determine the complete nucleotide sequence and conduct a phylogenetic analysis of genome variants of the Puumala virus isolated in the Saratov region., Materials and Methods: The samples for the study were field material collected in the Gagarinsky (formerly Saratovsky), Engelssky, Novoburassky and Khvalynsky districts of the Saratov region in the period from 2019 to 2022. To specifically enrich the Puumala virus genome in the samples, were used PCR and developed a specific primer panel. Next, the resulting PCR products were sequenced and the fragments were assembled into one sequence for each segment of the virus genome. To construct phylogenetic trees, the maximum parsimony algorithm was used., Results: Genetic variants of the Puumala virus isolated in the Saratov region have a high degree of genome similarity to each other, which indicates their unity of origin. According to phylogenetic analysis, they all form a separate branch in the cluster formed by hantaviruses from other subjects of the Volga Federal District. The virus variants from the Republics of Udmurtia and Tatarstan, as well as from the Samara and Ulyanovsk regions, are closest to the samples from the Saratov region., Conclusion: The data obtained show the presence of a pronounced territorial confinement of strains to certain regions or areas that are the natural biotopes of their carriers. This makes it possible to fairly accurately determine the territory of possible infection of patients and/or the circulation of carriers of these virus variants based on the sequence of individual segments of their genome.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus , the bank vole (45%); Apodemus flavicollis , the yellow-necked mouse (29%); and Apodemus agrarius , the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus , A. glareolus , and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis , one Mus musculus , and one Sorex minutus . We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus , 1 M. musculus , and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Puumala orthohantavirus (PUUV) has a wide distribution throughout Europe. Distinctive temporal patterns of spillover into the human population are related to population dynamics of the reservoir host, the bank vole ( Clethrionomys glareolus ). As the rodent host is tied to specific habitats with small individual ranges, PUUV genetic diversity is also highly correlated with geographic distance. Using sequenced portions of viral S and M segments, we determined whether geographic clusters were supported. Human cases of PUUV infections are concentrated in southeastern Austria. We detected four distinct genotypes: two genotypes of the Alpe-Adria (ALAD) lineage typically associated with southeast Europe, and two sublineages of the Central Europe (CE) lineage. One cluster of CE genotypes represents a phylogenetically distinct sublineage compared to previously reported CE clades, and extends the boundary of the CE lineage further south than previously reported.

Hantaviruses are zoonotic agents transmitted from small mammals, mainly rodents, to humans, where they provoke diseases such as Hemorrhagic fever with Renal Syndrome (HFRS) and its mild form, Nephropathia Epidemica (NE), or Hantavirus Cardio-Pulmonary Syndrome (HCPS). Hantaviruses are spread worldwide and monitoring animal reservoirs is of primary importance to control the zoonotic risk. Here, we describe the development of a pan-viral resequencing microarray (PathogenID v3.0) able to explore the genetic diversity of rodent-borne hantaviruses endemic in Europe. Among about 800 sequences tiled on the microarray, 52 correspond to a tight molecular sieve of hantavirus probes covering a large genetic landscape. RNAs from infected animal tissues or from laboratory strains have been reverse transcribed, amplified, then hybridized to the microarray. A classical BLASTN analysis applied to the sequence delivered through the microarray allows to identify the hantavirus species up to the exact geographical variant present in the tested samples. Geographical variants of the most common European hantaviruses from France, Germany, Slovenia and Finland, such as Puumala virus, Dobrava virus and Tula virus, were genetically discriminated. Furthermore, we precisely characterized geographical variants still unknown when the chip was conceived, such as Seoul virus isolates, recently emerged in France and the United Kingdom.

To screen diagnostic specimens for the presence of hantavirus genomes or to identify new hantaviruses in nature, the pan-hanta L-PCR assay, a broadly reactive nested reverse transcription polymerase chain reaction (RT-PCR) assay targeting the L segment, is highly preferred over other assays because of its universality and high sensitivity. In contrast, the geographic allocation of Puumala virus strains to defined outbreak regions in Germany was previously done based on S segment sequences. We show that the routinely generated partial L segment sequences resulting from the pan-hanta L-PCR assay provide sufficient phylogenetic signal to inform the molecular epidemiology of the Puumala virus. Consequently, an additional S segment analysis seems no longer necessary for the identification of the spatial origin of a virus strain., Competing Interests: The authors declare no conflict of interest.

Hantaviruses pose a public health concern worldwide causing haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV) is the most prevalent hantavirus in Central and Northern Europe, and causes a mild form of HFRS, also known as nephropathia epidemica (NE). In nature, the main host of PUUV is the bank vole (Myodes glareolus), and transmission to humans occurs through inhalation of aerosols from rodent excreta. Nephropathia epidemica is particularly prevalent in Nordic countries, however, few studies of PUUV have been performed in Norway. The aim of this study was to analyse the dynamics of PUUV in Norway and compare with bank vole population dynamics, and also to complement the current diagnostic methodology of NE in Norway. Our results showed a significant seasonal and geographical variation of NE, and a general parallel peak trend between bank vole population densities and human NE incidence. A real-time and a nested PCR were successfully established as an invaluable diagnostic tool, with detection and sequencing of PUUV in a human serum sample for the first time in Norway. Phylogenetic analysis showed clustering of the obtained human sample with previous Norwegian bank vole isolates., (© 2017 APMIS. Published by John Wiley & Sons Ltd.)

Puumala virus (PUUV) represents one of the most important hantaviruses in Central Europe. Phylogenetic analyses of PUUV strains indicate a strong genetic structuring of this hantavirus. Recently, PUUV sequences were identified in the natural reservoir, the bank vole (Myodes glareolus), collected in the northern part of Poland. The objective of this study was to evaluate the presence of PUUV in bank voles from southern Poland. A total of 72 bank voles were trapped in 2009 at six sites in this part of Poland. RT-PCR and IgG-ELISA analyses detected three PUUV positive voles at one trapping site. The PUUV-infected animals were identified by cytochrome b gene analysis to belong to the Carpathian and Eastern evolutionary lineages of bank vole. The novel PUUV S, M and L segment nucleotide sequences showed the closest similarity to sequences of the Russian PUUV lineage from Latvia, but were highly divergent to those previously found in northern Poland, Slovakia and Austria. In conclusion, the detection of a highly divergent PUUV lineage in southern Poland indicates the necessity of further bank vole monitoring in this region allowing rational public health measures to prevent human infections.

Puumala hantavirus (PUUV) is the most common and widespread hantavirus in Europe and is associated with a mild form of haemorrhagic fever with renal syndrome in humans, called nephropathia epidemica. This study presents the molecular characterization of PUUV circulating in bank voles in two regions of the Netherlands. Most human cases of hantavirus infection are from these two regions. Phylogenetic analysis of the (partial) S, M and L-segments indicated that the Dutch strains belong to the CE lineage, which includes PUUV strains from France, Germany and Belgium. We have identified two distinct groups of PUUV, corresponding with their geographic origin and with adjoining regions in neighbouring countries.

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.

Three species of Myodes voles known to harbor hantaviruses include the bank vole (Myodes glareolus), which serves as the reservoir host of Puumala virus (PUUV), the prototype arvicolid rodent-borne hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in Europe, and the grey red-backed vole (Myodes rufocanus) and royal vole (Myodes regulus) which carry two PUUV-like hantaviruses, designated Hokkaido virus (HOKV) and Muju virus (MUJV), respectively. To ascertain the hantavirus harbored by the northern red-backed vole (Myodes rutilus), we initially screened sera from 233 M. rutilus, as well as from 90 M. rufocanus and 110 M. glareolus, captured in western and eastern Siberia during June 2007 to October 2009, for anti-hantaviral antibodies. Thereafter, lung tissues from 44 seropositive voles were analyzed for hantavirus RNA by reverse transcription-polymerase chain reaction. Partial L-, M- and S-segment sequences, detected in M. rutilus and M. rufocanus, were closely related to HOKV, differing from previously published L-, M- and S-segment sequences of HOKV by 17.8-20.2%, 15.9-23.4% and 15.0-17.0% at the nucleotide level and 2.6-7.9%, 1.3-6.3% and 1.2-4.0% at the amino acid level, respectively. Alignment and comparison of hantavirus sequences from M. glareolus trapped in Tyumen Oblast showed very high sequence similarity to the Omsk lineage of PUUV. Phylogenetic analysis, using neighbor-joining, maximal likelihood and Bayesian methods, showed that HOKV strains shared a common ancestry with PUUV and exhibited geographic-specific clustering. This report provides the first molecular evidence that both M. rutilus and M. rufocanus harbor HOKV, which might represent a genetic variant of PUUV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3'-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.

Puumala virus (PUUV) causes mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS), and is responsible for the majority of hantavirus infections of humans in Fennoscandia, Central and Western Europe. Although there are relatively many PUUV sequences available from different European countries, little is known about the presence of this virus in Poland. During population studies in 2009 a total of 45 bank voles were trapped at three sites in north-eastern Poland, namely islands on Dejguny and Dobskie Lakes and in a forest near Mikołajki. S and M segment-specific RT-PCR assays detected PUUV RNA in three animals from the Mikołajki site. The obtained partial S and M segment sequences demonstrated the highest similarity to the corresponding segments of a PUUV strain from Latvia. Analysis of chest cavity fluid samples by IgG ELISA using a yeast-expressed PUUV nucleocapsid protein resulted in the detection of two seropositive samples, both being also RT-PCR positive. Interestingly, at the trapping site in Mikołajki PUUV-positive bank voles belong to the Carpathian and Eastern genetic lineages within this species. In conclusion, we herein present the first molecular evidence for PUUV in the rodent reservoir from Poland.

Puumala virus (PUUV) is considered a classic Old World etiologic agent of nephropathia epidemica (NE), or hemorrhagic fever with renal syndrome (HFRS). HFRS is considered to be distinct from hantavirus (cardio-)pulmonary syndrome (HPS or HCPS), described in the New World. Here, we report a severe case, which fulfilled most, if not all, Centers for Disease Control and Prevention (CDC) criteria for HPS, needing non-invasive ventilation and subsequent acute hemodialysis. However, the etiological agent was PUUV, as proved by serological testing, real-time polymerase chain reaction (PCR), and sequencing. Viral antigen was detected by specific anti-PUUV immunostaining, showing, for the first time, greenish intracytoplasmic inclusions in bronchoalveolar lavage (BAL) macrophages. This case definitely confirms that HPS can be encountered during PUUV infections. Interestingly, special findings could render the diagnosis easier, such as greenish homogeneous cytoplasmic inclusions, surrounded by a fine clear halo in BAL macrophages. Therefore, although the diagnosis remains difficult before the onset of renal involvement, the occurrence of severe respiratory failure mimicking community-acquired pneumonia must alert the clinician for possible HPS, especially in endemic areas.

Background: Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics., Objectives: This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses., Study Design: Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments., Results: By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV., Conclusions: This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Puumala virus causes nephropathia epidemica, a rodent-borne zoonosis that is endemic to Europe. We sequenced the complete Puumala virus genome that was directly recovered from a person who died and compared it with those of viruses from local bank voles. The virus strain involved was neither a unique nor rare genetic variant.

To investigate 2,017 cases of hantavirus disease in Germany, we compared 38 new patient-derived Puumala virus RNA sequences identified in 2010 with bank vole-derived small segment RNA sequences. The epidemic process was driven by outbreaks of 6 Puumala virus clades comprising strains of human and vole origin. Each clade corresponded to a different outbreak region.

Puumala hantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome in Europe. Seven genetic lineages of PUUV have thus far been recorded, which exhibit geographic structure within the distribution of its natural host, the bank vole (Myodes glareolus). This study presents evidence for two distinct PUUV lineages co-circulating in Latvia: one previously described from Russia and a novel one that appears to be endemic. The Latvian lineage (LAT) is considerably divergent and several amino acid markers make it easily distinguishable. Phylogenetic analysis suggested a possibility of different evolutionary histories for the PUUV genome segments of LAT., (Copyright © 2011 Wiley Periodicals, Inc.)

Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.

Hantaviruses, the causative agents of two emerging diseases, are negative-stranded RNA viruses with a tripartite genome. We isolated two substrains from a parental strain of Puumala hantavirus (PUUV-Pa), PUUV-small (PUUV-Sm) and PUUV-large (PUUV-La), named after their focus size when titrated. The two isolates were sequenced; this revealed differences at two positions in the nucleocapsid protein and two positions in the RNA-dependent RNA polymerase, but the glycoproteins were identical. We also detected a 43-nucleotide deletion in the PUUV-La S-segment 5' noncoding region covering a predicted hairpin loop structure that was found to be conserved among all hantaviruses with members of the rodent subfamily Arvicolinae as their hosts. Stocks of PUUV-La showed a lower ratio of viral RNA to infectious particles than stocks of PUUV-Sm and PUUV-Pa, indicating that PUUV-La replicated more efficiently in alpha/beta interferon (IFN-α/β)-defective Vero E6 cells. In Vero E6 cells, PUUV-La replicated to higher titers and PUUV-Sm replicated to lower titers than PUUV-Pa. In contrast, in IFN-competent MRC-5 cells, PUUV-La and PUUV-Sm replicated to similar levels, while PUUV-Pa progeny virus production was strongly inhibited. The different isolates clearly differed in their potential to induce innate immune responses in MRC-5 cells. PUUV-Pa caused stronger induction of IFN-β, ISG56, and MxA than PUUV-La and PUUV-Sm, while PUUV-Sm caused stronger MxA and ISG56 induction than PUUV-La. These data demonstrate that the phenotypes of isolated hantavirus substrains can have substantial differences compared to each other and to the parental strain. Importantly, this implies that the reported differences in phenotypes for hantaviruses might depend more on chance due to spontaneous mutations during passage than inherited true differences between hantaviruses.

The genus Hantavirus (family Bunyaviridae) includes negative-strand RNA viruses that are carried by persistently infected rodent and insectivore species. Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is a pathogenic hantavirus that causes outbreaks of mild haemorrhagic fever with renal syndrome across Europe. In northern Europe, PUUV is represented by several genetic lineages that are maintained by distinct phylogroups of bank voles. The present study describes sequences of new PUUV strains recovered from northern and southern regions of Scandinavia and compares phylogenetic relationships between north-European PUUV strains and M. glareolus. This analysis revealed contradictions in phylogenetic clustering and remarkable differences in estimated divergence times between the lineages of PUUV and its host, suggesting that the established PUUV lineages did not co-diverge with the distinct phylogroups of M. glareolus that carry them at present.

We report a case of hantavirus infection (nephropathia epidemica) diagnosed in a Belgian backpacker returning from a trekking expedition in Ecuador, after likely heavy exposure to rodents. Because of epidemiological inconsistency, molecular investigation was performed and revealed a Puumala infection acquired during very limited exposure in Belgium upon return.

In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjärvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32 % of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjärvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fennoscandia.

Serological and molecular evidence showed that a German female student became infected by Puumala hantavirus during mice trapping efforts in Finland. The incubation period before exhibiting clinical signs of nephropathia epidemica was as long as 6 weeks. Phylogenetic analysis of PUUV nucleic acid sequences amplified from the patient demonstrate that she was infected by the PUUV strain circulating at the place of her occupational exposure in Finland.

Hantavirus infections are known in Germany since the 1980s. While the overall antibody prevalence against hantaviruses in the general human population was estimated to be about 1-2%, an average of 100-200 clinical cases are recorded annually. In the years 2005 and 2007 in particular, a large increase of the number of human hantavirus infections in Germany was observed. The most affected regions were located in the federal states of Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, and Lower Saxony. In contrast to the well-documented situation in humans, the knowledge of the geographical distribution and frequency of hantavirus infections in their rodent reservoirs as well as any changes thereof was very limited. Hence, the network "Rodent-borne pathogens" was established in Germany allowing synergistic investigations of the rodent population dynamics, the prevalence and evolution of hantaviruses and other rodent-associated pathogens as well as their underlying mechanisms in order to understand their impact on the frequency of human infections. A monitoring of hantaviruses in rodents from endemic regions (Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, Lower Saxony) and regions with a low number of human cases (Mecklenburg Western-Pomerania, Brandenburg, Saxony, Saxony-Anhalt) was initiated. Within outbreak regions, a high prevalence of Puumala virus (PUUV) was detected in bank voles. Initial longitudinal studies in North Rhine Westphalia (city of Cologne), Bavaria (Lower Bavaria), and Lower Saxony (rural region close to Osnabrück) demonstrated a continuing presence of PUUV in the bank vole populations. These longitudinal studies will allow conclusions about the evolution of hantaviruses and other rodent-borne pathogens and changes in their distribution, which can be used for a risk assessment of human infections. This may become very important in order to evaluate changes in the epidemiology of rodent-borne pathogens in the light of expected global climate changes in the future.

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.

In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, "Bavaria," circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.

In order to detect the nucleic acid of Puumala hantavirus, RNA was extracted from lungs of bank voles captured in Northeast China, and partial S and M genome segments of Puumala virus were amplified by RT-PCR and sequenced. Phylogenetic analysis suggested that Chinese Puumala virus had diverged from the common node of PUUV, with accumulating nucleotide substitutions and formed a distinct lineage from other Puumala viruses. Newly found Puumala virus was most closely related to the Kamiiso-8Cr-95 and Tobetsu-60Cr-93 strains which came from Japan and the muju strains which came from South Korea. By analysis of S and M genome segments of Puumala virus, we deduced a new Puumala virus subtype did exist in Northeast China.

Phylogenetic relations were investigated in the group of Puumala viruses (the genus Hantavirus, the family Bunyaviridae) by the mutual information showing the similarity of the nucleotide sequences of S- and M-genome segments. There was evidence that recombination events might occurr in some Eurasian Puumala viruses (Scandinavian and West-Siberian genotypes). The investigation revealed the conservative sites available in the S and M segments that were common to most Puumala viruses, as well as the preservation of alternation and mutual arrangement of these sites in the S and M segments even for greatly diverging genotypes, which may be used in the detection of recombination events in Puumala viruses during further studies.

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.

Hantavirus serotype Puumala (PUUV) is the etiologic agent of Nephropathia epidemica (NE), a mild variant of Hemorrhagic Fever with Renal Syndrome (HFRS) in Europe, with lethality rates up to 1% among human patients. The serotype PUUV is composed of numerous geographically restricted strains forming a great number of phylogeographic lineages, sublineages, variants, and clusters. We describe a new, geographically and phylogenetically well-defined pathogenic PUUV sublineage in Southwest Germany (strain Heidelberg/hu) originated from an HFRS patient. The genetic analysis of PUUV strain Heidelberg/hu may contribute in future to better diagnostics, the development of vaccines, and the understanding of the factors that determine hantavirus pathogenesis.

Sequences of the Small (S) and the Medium (M) genome segments of Puumala hantavirus (PUUV) were recovered from bank voles Clethrionomys glareolus trapped at 2 locations, Klippitztörl (Carinthia) and Ernstbrunn (Lower Austria). Lung tissue samples from 12 rodents earlier found hantavirus antibody-positive were further screened for the presence of hantaviral N-antigen using immunoblotting. RNA purified from 7 N-Ag-positive samples was subjected to the reverse transcription-polymerase chain reaction with primers designed to recover the complete S segment sequence of PUUV. The amplicons of expected size (approximately 1800 bp) have been recovered from 2 samples, 1 from Klippitztörl and another from Ernstbrunn. From the same 2 samples, PCR amplicons corresponding to the partial M segment sequence (nt 2140 to 2594), have been prepared. The S and M amplicons were sequenced and subjected to genetic analyses. Recovered hantavirus genome sequences undoubtedly belonged to PUUV genotype and the corresponding wild-type hantavirus strains were designated as PUU/Klippitztörl/Cg9/1995 and PUU/Ernstbrunn/Cg641/1995. Phylogenetic analysis placed 2 Austrian PUUV strains within a distinct genetic lineage that also included strains from the neighbouring Slovenia and Croatia. This Alpe-Adrian lineage, previously designated as 'Balkan lineage', was well supported on both S and M phylogenetic trees.

Puumala virus is a member of the hantavirus genus in the Bunyaviridae family, and the major causative agent of haemorrhagic fever with renal syndrome in Europe. This study was conducted with a human Puumala virus isolate (PUUV Umeå/hu), and contains the determination of the first complete PUUV sequence from a human source. When the relationship to other Puumala viruses was analysed, a possible RNA segment exchange between two local strains of PUUV was noticed. Furthermore, the coding regions of PUUV Umeå/hu S- and M-segments were cloned, and a large set of gene products were expressed in mammalian cells. In addition, postulated N- and O-linked glycosylation sites in the two envelope proteins (Gn and Gc) were investigated individually by site-directed mutagenesis followed by gel-shift analysis. Our data demonstrate that N-linked glycosylation occurs at three sites in Gn (N142, N357 and N409), and at one site in Gc (N937). Also, one possible O-glycosylation site was identified in Gc (T985). We conclude that the diversity between different Puumala virus isolates is high, and consequently characterization of local PUUV isolates is important for clinical diagnostic work. Finally, the obtained results concerning the encoded gene products are of great importance for the design of new vaccines.

DNA microarrays combine high-precision technology with advanced molecular biology to achieve high-throughput screening of DNA fragments. In this study, we investigated the potential of the cDNA microarray technique to identify and discriminate PCR derived amplicons from genetically highly similar viruses. The wide range of sequence variation among hantaviruses makes them suitable as a model for this purpose. The hantaviruses, carried by rodents, cause several hundred thousand cases of severe human disease every year in many parts of the world. A hantavirus-specific microarray, including DNA fragments from 12 viral isolates of six different hantaviruses, was designed. The S and M genome segments were represented by 500-nucleotide overlapping and 250-nucleotide non-overlapping fragments. A considerable ability to distinguish between different hantaviruses was demonstrated using a novel analysis method. Even different isolates of a single virus, were identified correctly despite 90% sequence similarity. The distinction ability was accompanied by a tolerance for smaller sequence differences, which makes the microarray suitable for testing samples containing unknown viruses. Viral genetic material found in samples from the lungs of bank voles caught in the wild was identified precisely, which demonstrated further the potential for this technology., (Copyright 2004 Wiley-Liss, Inc.)

Hantaviruses belong to the Bunyaviridae family, which is comprised of Bunyavirus, Phlebovirus, Nairovirus, and Hantavirus. Euroasiatic Hantaviruses belong to two distinct subfamilies: Murinae (comprising Hantaan, Dobrava, and Seoul viruses), which are responsible for hemorrhagic fever with renal syndrome (HFRS), and Arvicolinae (comprising Puumala virus), responsible for nephropathia epidemica (NE) and HFRS. On the contrary, the New World Hantavirus belongs to the Sigmodontinae subfamily and includes the North American viruses: Sin Nombre, Monongahela; New York, Bayou, Black Creek Canal, and the South American, which comprise the Andes, Oran, Lechiguanas, Laguna Negra, Juquitiba; both groups are responsible for the hantavirus pulmonary syndrome (HPS).

In this study, two different hantaviruses, Puumala virus (PUUV) and Dobrava virus (DOBV), were demonstrated for the first time to coexist and cause hemorrhagic fever with renal syndrome (HFRS) in Croatia. Phylogenetic analysis showed some differences among the nucleotide sequences of PUUV originating from Dinara mountain, which was more closely related to Austrian PUUV than other Croatian PUUV from Mala Kapela mountain. More consistency was found among the Croatian DOBV. HFRS was verified in 85 of 201 suspected cases recorded in 1995 during the largest HFRS outbreak in Croatia. Most of these cases were soldiers. With the exception of the coastal region and islands, all of Croatia was found to be an area endemic for HFRS. A statistically significantly higher proportion of DOBV-infected patients had acute renal failure, visual disturbance, severe thrombocytopenia, and elevated levels of nonsegmented leukocytes, creatine, and total bilirubin. The prevalence of gastrointestinal and electrocardiography disorders also was greater in DOBV-infected patients. Interestingly, significantly more PUUV-infected patients had elevated systolic blood pressure on admission to the hospital. Further prospective studies are necessary to shed more light on differences in HFRS severity associated with PUU and DOB viruses., (Copyright 2002 Wiley-Liss, Inc.)

We report on the first Puumala hantavirus nucleotide sequence (strain Opina-916) amplified from a bank vole trapped in Slovakia, central Europe. Phylogenetic analysis of the S-segment sequence grouped the virus within the western/central European sublineage of Puumala virus. In the neighborhood of the rodent trapping site two cases of human infection by the Puumala virus were verified.