Genetic analyses of Seoul hantavirus genome recovered from rats (Rattus norvegicus) in the Netherlands unveils diverse routes of spread into Europe.

Seoul virus (SEOV) is the etiologic agent of hemorrhagic fever with renal syndrome. It is carried by brown rats (Rattus norvegicus), a commensal rodent that closely cohabitates with humans in urban environments. SEOV has a worldwide distribution, and in Europe, it has been found in rats in UK, France, Sweden, and Belgium, and human cases of SEOV infection have been reported in Germany, UK, France, and Belgium. In the search of hantaviruses in brown rats from the Netherlands, we found both serological and genetic evidence for the presence of SEOV in the local wild rat population. To further decipher the relationship with other SEOV variants globally, the complete genome of SEOV in the Netherlands was recovered. SEOV sequences obtained from three positive rats (captured at close trapping locations at the same time) were found highly similar. Phylogenetic analyses demonstrated that two lineages of SEOV circulate in Europe. Strains from the Netherlands and UK, together with the Baxter strain from US, constitute one of these two, while the second includes strains from Europe and Asia. Our results support a hypothesis of diverse routes of SEOV spread into Europe. These findings, combined with other indications on the expansion of the spatial European range of SEOV, suggest an increased risk of this virus for the public health, highlighting the need for increased surveillance.

Dobrava hantavirus variants found in Apodemus flavicollis mice in Kirklareli Province, Turkey.

Hantaviruses infect humans via inhalation of viral particles within secretions of infected rodents or rarely through direct contact with infected rodents. Determining the prevalence of hantavirus infections among rodent populations is of vital importance to obtain information on hantavirus-related cases and to predict possible outbreaks. We hypothesized that DOBV strains circulating in the Thrace Region in Turkey would be related to other Balkan DOBV strains. In this study, hantavirus infections in the rodent population of the Kirklareli-Igneada Region (north-western Turkey, near the Bulgarian border) were investigated. This region is of particular importance, as it is located in the south-eastern margin of the European continent and was used as an entrance point of Asian faunal elements into Europe. DOBV infection was detected in eight of 73 rodents; all were of the Apodemus flavicollis species. Partial sequences of the viral S-, M-, and L-genome segments were recovered and compared with previously reported DOBV sequences. The newly characterized Turkish strains were similar to other DOBV variants. Silent nucleotide mutations were dominant. The hantavirus prevalence in the Igneada region was similar to what has been reported in Greece and Bulgaria. For the first time, the M-segment sequences of DOBV from Turkey were recovered and genetic data of hantaviruses from Thrace region of Turkey were obtained.

Detection of Dobrava hantavirus RNA in Apodemus mice in Bulgaria.

Several Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Europe: Dobrava-Belgrade virus (DOBV), Puumala, Saaremaa, Sochi, and Seoul virus. Although HFRS is endemic in Bulgaria, genome sequences of hantaviruses have never been detected in wild rodents. To identify rodent reservoirs, a total of 691 rodents from three endemic regions were trapped in 2011-2012 and screened by TaqMan RT-PCR for detection of hantaviral genomic RNA. Partial small (S) and/or large (L)-segment sequences were recovered from six Apodemus mice: five of the species A. flavicollis and one A. agrarius. Phylogenetic analysis revealed that all recovered sequences belonged to DOBV. On the phylogenetic trees, the novel Bulgarian hantavirus sequences clustered together with sequences of established previously DOBV variants recovered from Bulgarian HFRS patients and also with variants found in wild rodents trapped in Slovenia, Greece, and Slovakia. One of the novel Bulgarian DOBV S-sequences from A. agrarius was related closely to DOBV sequences recovered from A. flavicollis, suggesting a spillover of DOBV from its natural host to A. agrarius mice. The results of this study confirmed the circulation of DOBV in wild rodents in Bulgaria. The complexity of the epidemiological situation in the Balkans requires further studies of hantaviruses in rodent hosts and human HFRS cases.

Analysis of complete Puumala virus genome, Finland.

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.

Co-circulation of two Puumala hantavirus lineages in Latvia: a Russian lineage described previously and a novel Latvian lineage.

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.

Genetic characterization of seoul hantavirus originated from norway rats (Rattus norvegicus) captured in Belgium.

Hantaviruses (genus Hantavirus, family Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. So far, in Europe, four pathogenic hantaviruses have been found, often in co-circulation: Puumala virus (PUUV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Seoul virus (SEOV). Of those, only PUUV was found in Belgium. Recently, in our search for hantaviruses in the Flanders region of Belgium we collected genetic and serological evidence for the presence of SEOV virus in local brown rats. In this article, the results of (phylo)genetic analysis of wild-type SEOV strain from the Flanders are presented. The analysis based on the complete S segment sequence and also partial M- and L-segment sequences revealed that the Belgian SEOV strain was related most closely to strains from France, Indonesia, Japan, Singapore, Cambodia (those associated with the species Rattus norvegicus) and Vietnam. Such a clustering was in perfect agreement with the results of direct sequence comparison and suggested the same evolutionary history for all three genome segments of the Belgian SEOV strain (i.e., no reassortment of genome segments). So far, SEOV has been found in two European countries, France and Belgium, and there is every reason to believe that the area of the virus distribution in Europe is not restricted to those countries.

Genetic evidence for the presence of two distinct hantaviruses associated with Apodemus mice in Croatia and analysis of local strains.

In Europe, Dobrava-Belgrade (DOBV), Saaremaa (SAAV), and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003-2004 were screened for the presence of hantaviral N-Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected-positive. Partial sequences of hantavirus M and S segments were recovered by RT-PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in Apodemus agrarius mouse, that is, not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases [Markotic et al., 2002] were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co-circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighboring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics.

Co-circulation of three pathogenic hantaviruses: Puumala, Dobrava, and Saaremaa in Hungary.

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow-necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well- supported Alpe-Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host-specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co-circulate in the same locality and can be maintained side-by-side in different rodent species.

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.

BACKGROUND: The 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. RESULTS: In 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). CONCLUSION: Our 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.

Genetic analysis of hantaviruses carried by Myodes and Microtus rodents in Buryatia.

Hantavirus genome sequences were recovered from tissue samples of Myodes rufocanus, Microtus fortis and Microtus oeconomus captured in the Baikal area of Buryatia, Russian Federation. Genetic analysis of S- and M-segment sequences of Buryatian hantavirus strains showed that Myodes-associated strains belong to Hokkaido virus (HOKV) type while Microtus-associated strains belong to Vladivostok virus (VLAV) type. On phylogenetic trees Buryatian HOKV strains were clustered together with M. rufocanus- originated strains from Japan, China and Far-East Russia (Primorsky region). Buryatian Microtus- originated strains shared a common recent ancestor with M. fortis- originated VLAV strain from Far-East Russia (Vladivostok area). Our data (i) confirm that M. rufocanus carries a hantavirus which is similar to but distinct from both Puumala virus carried by M. glareolus and Muju virus associated with M. regulus, (ii) confirm that M. fortis is the natural host for VLAV, and (iii) suggest M. oeconomus as an alternative host for VLAV.

Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered from patients and rodents.

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.

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.

Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand.

Sixty one tissue samples from several rodent species trapped in five provinces of Thailand were examined for the presence of hantaviral markers by enzyme-immunoassay and immunoblotting. Four samples, all from the great bandicoot rat Bandicota indica, were confirmed positive for the hantaviral N-antigen. Two of them were trapped in Nakhon Pathom province, the other two in Nakhon Ratchasima province, approximately 250 km from the other trapping site. When analysed by RT-nested PCR, all four rodents were found positive for the hantaviral S- and M-segment nucleotide sequences. Genetic analysis revealed that the four newly described wild-type strains belong to Thailand hantavirus. On the phylogenetic trees they formed a well-supported cluster within the group of Murinae-associated hantaviruses and shared a recent common ancestor with Seoul virus.

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.

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France.

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.

Hantavirus infections in Spain: analysis of sera from the general population and from patients with pneumonia, renal disease and hepatitis.

BACKGROUND: Hantaviruses are rodent borne viruses in the family Bunyaviridae that cause significant morbidity in large areas of Europe. There are only a few reports available on hantavirus infections from Spain. Although the results of these earlier studies indicated the presence of hantavirus infections, no confirmative or serotype-specific analyses have been performed. OBJECTIVES: To investigate whether hantaviruses cause human infection/disease in Spain. STUDY DESIGN: Ten thousand, four hundred and eighteen serum samples from the general population and 599 sera from 492 patients with potential hantavirus infections (renal disease, pneumonia or hepatitis) were initially screened by immunofluorescence assay (IFA) using Hantaan, Seoul and Puumala hantavirus antigens. Altogether 193 suspicious samples (165 from healthy people and 28 from patients) were selected for confirmation by quality-assured assays. RESULTS AND CONCLUSIONS: Of the 165 pre-screened serum samples from healthy individuals, only five could be confirmed by IFA for hantavirus-reactive antibodies (using Dobrava, Saaremaa, Hantaan or Puumala virus antigens). In addition, one serum was found weakly positive for hantavirus-reactive IgG by ELISA using recombinant Saaremaa virus (SAAV) nucleocapsid (N) antigen, and subsequently confirmed by immunoblotting. Thus, the results indicated a low (0.06%) total antibody prevalence to hantaviruses in Spain. Of 28 pre-screened serum samples from hospitalized patients, eight reacted as positive or showed border-line reactivities for hantavirus-specific IgM by ELISA using recombinant Saaremaa and Puumala virus N antigens. The IFA/ELISA reactive/border-line samples were subsequently analyzed by a focus reduction neutralization test, which revealed low titers (1:80) against SAAV in two samples from a patient with hepatic disease. The nature of the hantavirus(es) potentially involved remain, however, unknown, since none of the positive samples showed neutralizing titers of the expected range to any of the known European hantaviruses.

Distribution of Puumala hantavirus in Denmark: analysis of bank voles (Clethrionomys glareolus) from Fyn and Jutland.

The majority of hantaviral infections in Denmark since 1957 have occurred on the island of Fyn. We have recently shown the presence of Puumala hantavirus (PUUV) in bank voles (Clethrionomys glareolus) on Fyn. To learn more about the distribution and prevalence of PUUV in Denmark, 310 small mammals, belonging to six rodent and two insectivore species, were trapped in four localities on Fyn and three localities on the Jutland Peninsula (mainland Denmark), where only a few cases of nephropathia epidemica (NE) have been confirmed so far. Serum samples (heart extracts) from all animals were initially analyzed for the presence of hantavirus-specific antibodies (Ab) by enzyme immunoassay, and lung tissue samples of bank voles for the presence of the viral nucleocapsid protein antigen by immunoblotting. Altogether 11 Ab-positive rodents were found, all of them bank voles from Fyn. PUUV genome sequences (nucleotides 2,168-2,569 from the medium genomic segment) were recovered by reverse transcription-polymerase chain reaction from seven bank voles. They showed an overall diversity up to 5% and formed a well-supported genetic lineage on the phylogenetic tree with a clustering of two strains from southeastern Fyn together and a separation from the one strain from western Fyn. Our data confirmed the circulation of PUUV in both the southeastern and western parts of the island, known to have the highest numbers of human NE cases.

Microevolution of Puumala hantavirus during a complete population cycle of its host, the bank vole (Myodes glareolus).

Microevolution of Puumala hantavirus (PUUV) was studied throughout a population cycle of its host, the bank vole (Myodes glareolus). We monitored PUUV variants circulating in the host population in Central Finland over a five-year period that included two peak-phases and two population declines. Of 1369 bank voles examined, 360 (26.3%) were found infected with PUUV. Partial sequences of each of the three genome segments were recovered (approx. 12% of PUUV genome) from 356 bank voles. Analyses of these sequences disclosed the following features of PUUV evolution: 1) nucleotide substitutions are mostly silent and deduced amino acid changes are mainly conservative, suggesting stabilizing selection at the protein level; 2) the three genome segments accumulate mutations at a different rate; 3) some of the circulating PUUV variants are frequently observed while others are transient; 4) frequently occurring PUUV variants are composed of the most abundant segment genotypes (copious) and new transient variants are continually generated; 5) reassortment of PUUV genome segments occurs regularly and follows a specific pattern of segments association; 6) prevalence of reassortant variants oscillates with season and is higher in the autumn than in the spring; and 7) reassortants are transient, i.e., they are not competitively superior to their parental variants. Collectively, these observations support a quasi-neutral mode of PUUV microevolution with a steady generation of transient variants, including reassortants, and preservation of a few preferred genotypes.

A newly recognized hantavirus in the Asian house rat (Rattus tanezumi) in Indonesia.

Hantaviral sequences were recovered from the lung tissue of an Asian house rat (Rattus tanezumi) captured in Serang, Indonesia. Phylogenetic analysis of partial L, M and S segment sequences showed that they belonged to a novel hantavirus provisionally named Serang virus (SERV). Notably, SERV is distinct from the hantaviruses associated with rodents of the species Rattus: Seoul virus associated with Rattus norvegicus worldwide and Gou virus isolated from Rattus rattus in China. Instead SERV appeared more closely related to Thailand virus (THAIV) carried by the great bandicoot rat (Bandicota indica). These results suggest the possibility that SERV originated via host-switching, with a possible scenario of (pre)-THAIV 'jumping' from (pre)bandicoots to rats and colonizing this new host species.