VECTOR-BORNE AND ZOONOTIC DISEASES Volume 12, Number 6, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2011.0784

Tula Virus Infections in the Eurasian Water in Central Europe

Mathias Schlegel,1 Eveline Kindler,2 Sandra S. Essbauer,3 Ronny Wolf,4 Jo¨rg Thiel,5 Martin H. Groschup,1 Gerald Heckel,2,7 Rainer M. Oehme,6 and Rainer G. Ulrich1

Abstract

Recent reports of novel hantaviruses in shrews and moles and the detection of -borne hantaviruses in different rodent 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 arvalis, the East European vole M. levis (formerly rossiaemeridionalis), and subsequently in other Microtus species. Here we report the first multiple RT-PCR de- tection and sequence analyses of TULV in the Eurasian water vole 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 . 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 exper- imental infection studies have to prove the potential of A. amphibius as an additional TULV reservoir host.

Key Words: Arvicola amphibius—Central Europe—Hantavirus—Tula virus.

Introduction Kang et al. 2009). Transmission of hantaviruses is believed to be mainly indirect by inhalation of virus-contaminated rodent- antaviruses represent a of the family Bu- excreta derived aerosols, whereas bites seem to represent a Hnyaviridae with emerging human pathogenic represen- very rare mode of transmission (Scho¨nrich et al. 2008). tatives in Europe, Asia, and the Americas. Despite the problem On the Eurasian continent hantaviruses may cause of isolating and propagating these viruses, in the last three hemorrhagic fever with renal syndrome (HFRS) of different decades the knowledge of European and Asian hantaviruses severity and case fatality rates of up to 10% (Kru¨ ger et al. has broadened significantly. Thus many new hantavirus spe- 2011). One representative is the bank vole Myodes glareolus- cies and strains, their host association, and prevalence and the transmitted Puumala virus (PUUV) distributed in almost all frequency of human infection and disease have been charac- parts of Europe and causing a mild to moderate form of HFRS, terized. Most importantly, besides the rodent-borne hanta- designated nephropathia epidemica, with a case fatality viruses during recent years a large number of novel rate of < 0.1% (Brummer-Korvenkontio et al. 1999). Second, hantaviruses were detected in different shrew and mole species Dobrava-Belgrade virus (DOBV) genetic lineages DOBV- in Europe and Asia, with thus far unknown human pathoge- Af, DOBV-Ap, DOBV-Aa, and Saaremaa associated with the nicity (Song et al. 2007a,2007b,2007c,2009; Arai et al. 2008; yellow-necked mouse Apodemus flavicollis, Caucasian wood

1Friedrich-Loeffler-Institut, Institute for Novel and Emerging Infectious Diseases, Greifswald–Insel Riems, Germany. 2Computational and Molecular Population Genetics (CMPG), Institute of Ecology and Evolution, University of Bern, Bern, Switzerland. 3Bundeswehr Institute of Microbiology, Department of Virology and Rickettsiology, Munich, Germany. 4Institute for Biology, University of Leipzig, Leipzig, Germany. 5Thu¨ ringer Landesanstalt fu¨ r Wald, Jagd und Fischerei, Gotha, Germany. 6Landesgesundheitsamt Baden-Wuerttemberg, Stuttgart, Germany. 7Swiss Institute of Bioinformatics, Genopode, Lausanne, Switzerland.

503 504 SCHLEGEL ET AL. mouse A. ponticus, or striped field mouse A. agrarius cause trapped at three sites in Germany were included in this study more severe, moderate, or mild/moderate cases of HFRS in (Fig. 1). The were necropsied according to standard certain parts of Europe, respectively (Avsic-Zupanc et al. protocols (Ulrich et al. 2008). 1999; Sibold et al. 2001; Vapalahti et al. 2003; Golovljova et al. 2007; Klempa et al. 2008; Kru¨ ger et al. 2011). RT-PCR and sequencing The knowledge of the human pathogenicity of the third Lung samples were investigated by two L-, one S-, and one hantavirus in Europe, the Tula virus (TULV), is sparse; there M-segment-specific RT-PCR assays. The initial screening oc- are only few reports of human TULV infections (Vapalahti curred through a newly established SYBR-Green-based real- et al. 1996; Schultze et al. 2002; Clement et al. 2003; Klempa time reverse transcription-PCR (RT-qPCR) assay using the et al. 2003; Mertens et al. 2011). This hantavirus has initially QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Ger- been found in the common vole Microtus arvalis, the East many) and novel degenerated primers (L2797F 5¢ GAR GAR European vole M. levis (formerly rossiaemeridionalis), and TAY ATH TCN TAT GGD GG-3¢; L2951R 5¢-HGG NGA CCA subsequently in M. agrestis, M. subterraneus, and M. gregalis, YTT NGT DGC AT-3¢) targeting a conserved region in the and is broadly distributed in Central Europe (Plyusnin et al. L-segment (nt 2797-2819 and nt 2951-2970; positions accord- 1994; Sibold et al. 1995; Song et al., 2002; Scharninghausen ing to NCBI reference sequence TULV, accession number: NC et al., 2002; Schmidt-Chanasit et al. 2010). 005226) of different rodent- and insectivore-borne Old World In general, the genome of hantaviruses is represented by hantaviruses. This assay was experimentally shown to detect three single-stranded RNA genome segments of negative DOBV-, PUUV-, and TULV-specific nucleic acid sequences in polarity. The structural proteins of the hantaviruses, the nu- lungs from naturally-infected Apodemus, Myodes, and Micro- cleocapsid (N) protein, and the glycoproteins Gn (G1) and Gc tus from Germany (data not shown). Each water vole (G2), are encoded by the small (S) segment of 1.6–2.0 kilobases lung sample was determined as positive if the ct-value was (kb), and the medium (M) segment of 3.5–3.6 kb, respectively. < 38 and by detection of a specific amplification product in a The RNA-dependent RNA polymerase is encoded by the melting curve and in a 1.5% agarose gel. Samples with no ct- large (L) segment of approximately 6.5 kb. value and no specific amplification product were defined as The close association of a single hantavirus species with a negative, whereas those with a ct-value > 38 and a typical single reservoir or closely related species of the same genus melting curve were considered as equivocal. In addition, the has been explained by a co-evolution hypothesis (Plyusnin samples from Baden-Wuerttemberg were tested in a nested and Morzunov 2001). However, the increasing number of RT-PCR assay using S-segment-specific primers (Sibold et al. hantavirus species, hantavirus studies on sympatrically- 1999). All RT-qPCR positive and equivocal samples, as well as occurring rodent reservoir species (Schmidt-Chanasit et al. positive samples from the nested RT-PCR assay, were also

2010), and the discovery of insectivore-borne hantaviruses in tested in a One-Step RT-PCR assay using a Superscript III One particular raises major questions on the evolution and host Step RT-PCR Kit (Invitrogen, Darmstadt, Germany), and with adaptation of hantavirus species (Henttonen et al. 2008). Al- primers targeting another region in the L-segment (Klempa ternatively to the virus-host co-evolution, recent studies have et al. 2006). Thereafter, all L-segment-positive lung RNA postulated a scenario of host switching and local host-specific samples were tested in S- and M-segment RT-PCR assays adaptation for hantavirus/host evolution (Ramsden et al. using primers specific for TULV and PUUV (Essbauer et al. 2008). Further, a host switch event in the distant past has been 2006; Schmidt-Chanasit et al. 2010). The RT-PCR products postulated for the ancestor of the -associated were purified with a PCR Purification Kit (Qiagen), and Khabarovsk virus (Vapalahti et al. 1999). sequenced using the BigDye terminator sequencing kit The Eurasian water vole (Arvicola amphibius, formerly (Perkin-Elmer, Waltham, MA) on an ABI 310 Genetic Analy- A. terrestris), like Microtus, is a member of the subfamily Ar- zer (Applied Biosystems, Foster City, CA). vicolinae, and is widely distributed in Europe. Interactions or sympatric occurrences of water voles with other arvicolines Phylogenetic analysis (e.g., M. arvalis) have been described, and their fluctuations in the population density sometimes correlate with the popula- The phylogenetic analyses were performed using MrBayes tion density in A. amphibius (Wieland 1973). So far investiga- 3.1.2 (Ronquist and Huelsenbeck 2003) with Bayesian tions of hantavirus infections in A. amphibius are sparse. Metropolis-Hastings Markov Chain Monte Carlo (MCMC) Hantavirus antigen was detected in lung samples of A. am- tree-sampling methods based on two MCMC runs consisting phibius from Russia using immunoglobulin G (IgG) antibodies of four chains of 2,000,000 generations with a burn-in of 25%, directed against PUUV, Hantaan virus, and Vladivostok virus and second by maximum-likelihood (ML) analysis calculated (Butenko et al. 1997). PUUV-reactive antibodies were demon- on a web server (http://www.phylo.org/sub_sections/ strated in 5.5% of 164 montane water voles (Arvicola scherman) portal). The optimal nucleotide substitution models according trapped in France (Charbonnel et al. 2008). In this study, we to jModeltest (Posada 2008) were the Transition Model 3 report the first molecular evidence of multiple TULV infections (TIM3) with a proportion of invariable sites (I), and a gamma- in A. amphibius from Germany and Switzerland. shaped distribution (G) for the L-segment, the Hasegawa, Kishino, and Yano Model (HKY) + I + G for the M-segment, Materials and Methods and the Transition Model 2 (TIM2) + G for the S-segment sequences. All partial L-, S-, and M-segment sequences were Rodent trapping and necropsy screened for recombination using the program RDP3 (Martin During 2001 to 2009 a total of 424 A. amphibius were trap- et al. 2010), which comprises six recombination detection ped at 20 different sites in Germany and at one locality in approaches (Bootscan, Chimeric, GENECONV, MaxChi, Switzerland. In addition, six M. arvalis and one M. agrestis RDP, and SiScan) with their default parameters. TULA VIRUS INFECTIONS IN THE EURASIAN WATER VOLE 505

FIG. 1. Map of trapping sites with RT-PCR/serologically positive and negative Arvicola amphibius, Microtus arvalis, and M. agrestis (stars indicate trapping sites with seroreactive and RT-PCR positive/equivocal A. amphibius; circles with white centers indicate trapping sites with seroreactive but RT-PCR-negative A. amphibius; diamonds indicate trapping sites with RT-PCR equivocal and negative A. amphibius; circles with crosses in the center indicate trapping sites with RT-PCR negative A. amphibius; solid circles indicate trapping sites with Tula virus (TULV)-RT-PCR-positive M. arvalis and M. agrestis; ‘‘x’’ indicates the origin of published TULV sequences; Schmidt-Chanasit, et al. 2010). Trapping sites: Ben, Bendelin; Ebe, Eberswalde; Mrz, Marzehns; Bieb, Biebersdorf; Duer, Du¨ rrweitzschen; Schar, Scharfenberg; Goett, Go¨ettingen; Sen, Sen- nickerode; Wen, Wenigenlupnitz; Sieb, Siebleben; Eck, Eckhardtshausen; Graf, Grafenwo¨hr; Winn, Winnenden; Wies, Wie- sensteig; Roem, Ro¨merstein; Heu, Heuberg; Nof, Noflen.

Serology BLAST search-mediated comparison of the novel cyt b se- quences with sequences available in GenBank (http:// Serological screening of phosphate-buffered saline (PBS) www.ncbi.nlm.nih.gov). Phylogenetic analyses of cyt b se- diluted chest cavity fluid (CCF) was done by an in-house IgG quences were performed as described for sequences derived ELISA using a yeast-expressed TULV nucleocapsid protein from TULV. The optimal nucleotide substitution model was (Mertens et al. 2011), and by following a previously published HKY + G. Sequences from the species Arvicola sapidus, as well protocol (Essbauer et al. 2006). The CCFs were diluted 1:10 in as additional sequences from A. amphibius available in Gen- 0.5% bovine serum albumin/0.05% Tween-20. Horseradish Bank were included in the analysis. The phylogenetic tree was peroxidase (HRP)-conjugated goat anti-mouse IgG (Bio-Rad, rooted with a sequence from Myodes glareolus as an outgroup. Hercules, CA) was used as secondary antibody. Detection of specific antibodies was accomplished by addition of 3,3¢,5,5’-Tetramethylbenzidine (TMB) substrate (Bio-Rad). Results The reaction was stopped by the addition of 50 lLof1M An initial screening of all 424 Arvicola lung samples with H SO (10 min). 2 4 the new SYBR-Green RT-qPCR assay identified 4 positive and 32 equivocal samples from eight different trapping sites in Cytochrome b PCR Germany and Switzerland (Table 1 and Fig. 1). The subse- For all TULV-RT-PCR-positive A. amphibius from the seven quent analysis of all 36 RT-qPCR-positive and equivocal trapping sites and one additional non-infected individual samples and 2 RT-qPCR-negative, but previously positive from each trapping site the morphological species determi- screened samples (GER127 and GER129) in a One-Step RT- nation was confirmed by a mitochondrial cytochrome b (cyt b) PCR assay with primers targeting another region in the gene-specific PCR (Schlegel et al. 2011), and a subsequent L-segment (Klempa et al. 2006) revealed a total of 8 samples

Table 1. List of RT-PCR- and/or Serologically-Positive Arvicola amphibius

RT-PCR

Rodent SYBR-Green One step One step One step real-time L-segment RT-PCR RT-PCR RT-PCR Country and region District Trapping site Number Sex TULV-IgG ELISAb (ct-value) L-segment S-segment M-segment

Switzerland Canton of Bern Bern-Mittelland Nof CH/09/1026 F +++ 28.48 Positive Positive Positive Germany Saxony Meißen Schar GER/08/712 F +++ 33.76 Positive Positive Positive GER/08/725 F ( + ) Neg Neg ND ND GER/08/738 F + Neg Neg ND ND

506 Leipzig Duer GER/09/815 M +++ 37.10 Positive Positive Positive Germany Thuringia Wartburgkreis Wen GER/09/21-00,30,35,36a M(+ ) Neg Neg ND ND GER/09/21-08,23a F(+ ) Neg Neg ND ND GER/09/21-02,19,20,26a F + Neg Neg ND ND Eck GER/09/2155 M +++ 37.09 Positive Positive Positive Germany Baden-Wuerttemberg Reutlingen Roem GER/109 F ND 38.02 Positive Positive Positivec Go¨eppingen Wies GER/127 M ND Neg Positive Positive Positive GER/139 F ND Neg Positivec Neg Positive Rems-Murr Winn GER/152 M ND 40.81 Positive Positive Positive

aSamples with the same ELISA reactivity and RT-PCR results. bOptical density values: +++, ‡ 2.8; + , 0.5–0.3; ( + ), 0.2–0.13; neg, < lower cut-off (on average 0.041). cAgarose gel analysis showed a weak amplification product of the expected size; sequencing approaches remained unsuccessful. F, female; M, male; ND, not done; Neg, negative; TULV, Tula virus; Duer, Du¨ rrweitzschen; Wen, Wenigenlupnitz; Roem, Ro¨merstein; Winn, Winnenden; Wies, Wiesensteig; Schar, Scharfenberg; Nof, Noflen; Eck, Eckhardtshausen. TULA VIRUS INFECTIONS IN THE EURASIAN WATER VOLE 507 showing the expected amplification product in the agarose Discussion gel, but only for 7 samples could a sequence be obtained (Table 1). These sequences represent a 336-bp-long part of the In this study we report the first multiple molecular evi- L-segment (nt 2962–3297 encoding aa 988–1099 of the RNA dence of Microtus-associated TULV infections in a represen- polymerase). tative of another genus of the subfamily Arvicolinae (i.e., in An initial BLAST search demonstrated the strongest simi- the Eurasian water vole). This finding is based on sequences larity of these Arvicola-derived L-segment sequences to cor- from all three TULV genome segments from a large panel of responding TULV sequences from M. arvalis and M. agrestis water vole samples from different geographic regions in (data not shown). Therefore, we determined additional novel Germany and Switzerland. The observed frequent spillover TULV partial L-segment as well as partial S- and M-segment (or host switch) is highly unexpected, as paleozoological and sequences from M. arvalis and M. agrestis trapped in Baden- molecular investigations suggest a last common ancestor of Wuerttemberg (Heu), Brandenburg (Bieb), and Thuringia Microtus and Arvicola more than three million years ago (Sieb) near sites where positive A. amphibius have been (Chaline and Graf 1988; Conroy and Cook 1999; Abramson previously identified (Fig. 1). A pair-wise comparison of the et al. 2009; Fink et al. 2010). Such transmissions of a hantavirus Arvicola-derived TULV L-segment and deduced RNA poly- from the original reservoir host to distantly related species merase sequences revealed nucleotide and amino acid se- other than humans have been reported only very rarely in quence differences of 14.6–21% and 0–5.5%, respectively. All 8 Europe, as for example a single house mouse infected by L-segment positive lung RNA samples were then tested in Apodemus flavicollis-associated Dobrava-Belgrade virus S- and M-segment RT-PCR assays using TULV- and PUUV- (Weidmann et al. 2005). Spillover infections have been specific primers. The obtained RT-PCR products were documented in numerous studies in the New World, with sequenced and found to represent a 378-bp-long part of the detection of Microtus-borne viruses in Peromyscus and Sig- M-segment (nt 2365–2742 encoding aa 789–914 of the glyco- modon species, Peromyscus-borne viruses in Reithrodontomys protein precursor), and a 549-bp-long part of the S-segment species, chipmunks and Mus species, Reithrodontomys-borne (nt 379–927 encoding aa 127–305 of the N protein). viruses in voles and wood rats, and Oryzomys-borne viruses The phylogenetic analyses of these partial L-, S-, and in Sigmodon species (Ulrich et al. 2002). M-segment sequences showed that the novel A. amphibius- The geographical clustering of the Arvicola-borne se- derived sequences cluster geographically with TULV se- quences with Microtus-derived TULV sequences from differ- quences derived from M. arvalis and M. agrestis in Germany ent parts of Germany and Europe suggests multiple spillover and Europe, but are clearly separated from sequences of infections of TULV from M. arvalis or M. agrestis to A. am- Microtus-borne Prospect Hill virus and Isla Vista virus and phibius. Although an opposite direction of the spillover in-

other Arvicolinae-derived hantaviruses (Fig. 2A, B, and C). fections seems possible, the frequency of molecular detection All TULV S- and M-segment sequences from Arvicola are part of TULV in Microtus species, however, suggests that members of two well-supported groups ‘‘Germany I’’ and ‘‘Germany of this genus represent the reservoir host (Plyusnin et al. 1994; III/Switzerland.’’ For both segments, the first group contains Sibold et al. 1995; Schmidt-Chanasit et al. 2010). In addition, sequences derived from A. amphibius from the federal states the detection of TULV-reactive antibodies without detection Thuringia and Saxony (trapping sites Eck, Schar, and Duer), of TULV-specific RNA in the water vole may also support this clustering together with TULV sequences from M. arvalis conclusion. Although the route of TULV transmission from (Marv) and M. agrestis (Magr) from Lower Saxony (trapping Microtus to Arvicola is not known, both species are sometimes sites Sen and Goett), Thuringia (trapping site Sieb), and found in the same habitats and may even use the same bur- Brandenburg (trapping sites Bieb, Mrz, Ben, and Ebe) (Fig. 2B rows (G Heckel, R Wolf, personal communication). and C). The group ‘‘Germany III/Switzerland’’ comprises Alternatively, the multiple molecular detection of TULV water vole-derived TULV sequences from Baden-Wuerttem- infections in water voles from several sites and different berg (trapping sites Roem, Winn, and Wies), and Switzerland geographic regions in Germany and Switzerland indicates the (trapping site Nof), as well as Microtus-derived sequences potential of this rodent species as an additional reservoir host from Baden-Wuerttemberg (trapping site Heu), and Bavaria of this particular virus. Thus our findings also raise more (trapping site Graf) (Fig. 2B and C). Pair-wise comparisons of general questions on the definition of a reservoir host for a the novel partial Arvicola-derived TULV S- and M-segment given hantavirus and the role of hantavirus/rodent co- nucleotide sequences (and deduced amino acid sequences) speciation in their molecular evolution. Usually the multiple showed divergences of 0.2–20.4% (0–8.4%) and 0.3–19.9% (0– detection of nucleic acid sequences in a single reservoir host 1.6%), respectively (data not shown). The screening for re- and their absence in sympatrically-occurring other rodent or combination did not detect any putative recombinant regions. small species is believed to be indicative of a reser- The serological screening of 286 A. amphibius from 5 dif- voir host function (Hjelle and Yates 2001). Thus, M. arvalis has ferent trapping sites where CCF was available identified 16 initially been identified as the most likely reservoir host of reactive samples with prevalences of 2.3–27% (Fig. 1 and TULV (Plyusnin et al. 1994; Sibold et al. 1995). However, Table 1). TULV was molecularly detected in additional Microtus spe- Phylogenetic analysis of cyt b sequences did not support cies (i.e., M. levis, formerly M. rossiaemeridionalis), M. agrestis, evolutionary distinctness of the novel A. amphibius sequences M. subterraneus, and M. gregalis (Plyusnin et al. 1994; Sibold from Germany and Switzerland, but rather showed relatively et al. 1995; Schmidt-Chanasit et al. 2010), and as here de- high similarity with other A. amphibius sequences from Swit- scribed in A. amphibius. Similarly, a TULV infection has been zerland and Finland (Fig. 3). The cyt b sequences derived from recorded in lagurus, a representative from another Spanish southern water voles A. sapidus formed an own genus of the Arvicolinae subfamily (GenBank accession cluster. numbers AF442619 and AF442618; Dekonenko and

FIG. 2. Bayesian trees based on partial L-segment (336 nucleotides) (A), S-segment (549 nucleotides) (B), and M-segment (378 nucleotides) (C) nucleotide sequences of the novel Arvicola- and Microtus-associated Tula virus (TULV) sequences, Microtus-associated Prospect Hill virus and Isla Vista virus sequences, and other hantavirus sequences. Posterior probabilities for Bayesian analysis are given before the slashes, and aLRT (approximate Likelihood-Ratio Test) values for branches for maximum likelihood (ML) analysis are shown after the slashes. Only values ‡ 0.7% and ‡ 70% are shown (values £ 0.7% and £ 70% are indicated by hyphens; scale bar indicates the number of nucleotide substitutions per site; asterisks indicate differences in the topology between ML- and Bayesian-tree at this node). Novel TULV sequences derived from A. amphibius, M. arvalis, and M. agrestis are highlighted by gray boxes. Outgroup clusters of closely-related sequences were collapsed (B). Outgroup cluster accession numbers: Isla Vista virus (U31535, U31534, and U19302); Puumala virus (NC_005224, Z84204, and AY526219); Khabarovsk virus (EU360900, EU360897, and EU360898); Yakeshi virus (EU072484 and EU072482); Yuan- jiang virus (FJ170795, FJ170793, FJ170794, and FJ170792); Shenyang virus (FJ170797 and FJ170796); Fusong/Vladivostok virus (EU072481, AB011630, and EU072480); Marv, Microtus arvalis; Magr, Microtus agrestis; Aamp, Arvicola amphibius; Llag, La- gurus lagurus; Mgre, Microtus gregalis; Mros, Microtus levis (formerly rossiaemeridionalis); Msub, Microtus subterraneus, Mmax, Microtus maximowiczii; Mfor, Microtus fortis; V, virus cell culture isolate.

508 TULA VIRUS INFECTIONS IN THE EURASIAN WATER VOLE 509

FIG. 2. (Continued). 510 SCHLEGEL ET AL.

FIG. 2. (Continued).

Yakimenko, unpublished data). Moreover, a previous study Baden-Wuerttemberg, southeast and southwest Germany, suggested an already established isolated replication and thus enlarges our knowledge of the geographical distribution transmission cycle of TULV in M. agrestis (Schmidt-Chanasit of this virus. Obviously, this virus has a broad, likely et al. 2010). Finally, these findings are in contrast to the usually Germany-wide, distribution. Moreover, the detection of a assumed co-evolution hypothesis, according to which one TULV sequence in Arvicola from Switzerland is the first mo- would expect different TULV lineages in Arvicola and Micro- lecular detection of this virus in Switzerland, as previously tus reservoirs. only a clinical case caused by TULV was reported, using focus TULV has previously been detected in reservoir hosts from reduction neutralization assay analysis (Schultze et al. 2002). several parts of Germany and from Russia, Slovakia, Croatia, This broad geographical distribution of TULV in the res- the Czech Republic, Austria, Poland, Belgium, France, ervoir host also raises questions about the reasons for the Hungary, The Netherlands, and Slovenia (Schmidt-Chanasit currently very low number of reported human TULV infec- et al. 2010). The herein described detection of TULV sequences tions, with only two case reports of potential TULV-induced in A. amphibius, M. arvalis, and M. agrestis from Thuringia and disease in humans (Schultze et al. 2002; Clement et al. 2003; TULA VIRUS INFECTIONS IN THE EURASIAN WATER VOLE 511

FIG. 3. Bayesian tree based on partial cytochrome b sequences (608 bp) of Tula virus-RT-PCR-positive (*) and negative Arvicola amphibius from Germany and Switzerland. Posterior probabilities for Bayesian analysis are given before the slashes, and aLRT (approximate Likelihood-Ratio Test) values for branches for maximum likelihood analysis after the slashes. Only values ‡ 0.7% and ‡ 70% are shown; values £ 0.7% and £ 70% are indicated by hyphens. Scale bar indicates the number of nucleotide substitutions per site. The outgroup branch is condensed and the divergence to Arvicola spp. is given in per- centages at the branches in the tree. The novel sequences are shown in bold. Sequences from the sister species A. sapidus, and additional sequences from A. amphibius available from GenBank were included, and their accession numbers are displayed in the tree. Myodes glareolus was used as an outgroup to root the phylogenetic tree.

Klempa et al. 2003). TULV-related Microtus-associated han- virus to infect distantly-related rodents of two different gen- taviruses in the New World (i.e., Prospect Hill virus, Blood- era. Future studies of wild water voles and other Arvicola land Lake virus, and Isla Vista virus) have not been shown to species from different regions in Europe are needed to verify cause significant disease in humans (Lee et al. 1985; Hjelle the frequency of spillover and/or host-switch events for et al. 1995; Song et al. 1995). Similarly, TULV and another TULV, and may thus allow the definition of the host range of Eurasian hantavirus species (Khabarovsk virus hosted by the TULV. These studies should be accompanied by experimental reed vole Microtus fortis) (Ho¨rling et al. 1996), are believed to infection studies addressing the pathogenic consequences of have low or no pathogenicity for humans. The low frequency TULV infections in different hosts, and the viral and host of the detection of human TULV infections might be ex- factors determining the host range, transmission pathways, plained by its low pathogenic potential as determined in cell and human pathogenicity of this virus. culture experiments (Kraus et al. 2004). On the other hand, a recent study in a forestry worker risk group in a region where Acknowledgments TULV has been detected in Microtus reservoirs demonstrated frequent serological detection of TULV-reactive antibodies in The authors kindly acknowledge the support of Do¨rte an ELISA test using the homologous antigen (Mertens et al. Kaufmann, Astrid Thomas, Kathrin Hirsbrunner, Cornelia 2011). Therefore, more extended serological investigations Triebenbacher, Anja Globig, Gerhard Dobler, Daniel Windolph, should determine if TULV represents a neglected human Michael Noack, Paul-Walter Lo¨hr, Johannes Lang, Thomas pathogenic hantavirus that is currently overlooked in human Schro¨der, Dietrich Heidecke, Jens Jacob, Hans-Joachim Pelz, infections due to the use of insufficient diagnostic tools. Thorsten Menke, Hermann Ansorge, Denny Maaz, Matthias In conclusion, multiple molecular detections of TULV in- Tzschoppe, Lutz-Florian Otto, Martin Kaatz, as well as the fections in water voles underline the unique potential of this helpful comments from Lutz C. Maul. 512 SCHLEGEL ET AL.

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