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Bat-associated hantaviruses have been detected in Asia, Africa and Europe. Recently, a novel hantavirus (Brno loanvirus, BRNV) was identified in common noctule bats (Nyctalus noctula) in the Czech Republic, but nothing is known about its geographical range and prevalence. The objective of this study was to evaluate the distribution and host specificity of BRNV by testing bats from neighbouring countries Germany, Austria and Poland. One thousand forty-seven bats representing 21 species from Germany, 464 bats representing 18 species from Austria and 77 bats representing 12 species from Poland were screened by L segment broad-spectrum nested reverse transcription-polymerase chain reaction (RT-PCR) or by BRNV-specific real-time RT-PCR. Three common noctules from Germany, one common noctule from Austria and three common noctules from Poland were positive in the hantavirus RNA screening. Conventional RT-PCR and primer walking resulted in the amplification of partial L segment and (almost) complete S and M segment coding sequences for samples from Germany and partial L segment sequences for samples from Poland. Phylogenetic analysis of these nucleotide sequences showed highest similarity to BRNV from Czech Republic. The exclusive detection of BRNV in common noctules from different countries suggests high host specificity. The RNA detection rate in common noctules ranged between 1 of 207 (0.5%; Austria), 3 of 245 (1.2%; Germany) and 3 of 20 (15%; Poland). In conclusion, this study demonstrates a broader distribution of BRNV in common noctules in Central Europe, but at low to moderate prevalence. Additional studies are needed to prove the zoonotic potential of this hantavirus and evaluate its transmission within bat populations., (© 2022. The Author(s).)

The striped field mouse (Apodemus agrarius) is known to carry several zoonotic pathogens, including Leptospira spp. and Dobrava-Belgrade orthohantavirus (DOBV). Since its first detection in 1996 in south-east Austria, the striped field mouse has further expanded its range in Austria. Here, we screened 35 striped field mice collected in an Austrian region near the Hungarian border for DOBV, Leptospira spp. and seven vector-borne pathogens. Hantavirus RT-PCR screening and DOBV IgG ELISA analysis led to the detection of two DOBV-positive striped field mice. The complete coding sequences of all three genome segments of both strains were determined by a combination of target enrichment and next-generation sequencing. Both complete coding S segment sequences clustered within the DOBV genotype Kurkino clade with the highest similarity to a sequence from Hungary. In one of 35 striped field mice, Leptospira borgpetersenii sequence type (ST) 146 was detected. Bartonella spp., Borrelia miyamotoi and Neoehrlichia mikurensis DNA was detected in four, one and two of 32 mice, respectively. Babesia, Anaplasma, Ehrlichia and Rickettsia specific DNA was not detected. Future investigations will have to determine the prevalence and invasion of these pathogens with the ongoing range expansion of the striped field mouse in Austria., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The natural host ranges of many viruses are restricted to very specific taxa. Little is known about the molecular barriers between species that lead to the establishment of this restriction or generally prevent virus emergence in new hosts. Here, we identify genomic polymorphisms in a natural rodent host associated with a strong genetic barrier to the transmission of European Tula orthohantavirus (TULV). We analysed the very abrupt spatial transition between two major phylogenetic clades in TULV across the comparatively much wider natural hybrid zone between evolutionary lineages of their reservoir host, the common vole (Microtus arvalis). Genomic scans of 79,225 single nucleotide polymorphisms (SNPs) in 323 TULV-infected host individuals detected 30 SNPs that were consistently associated with the TULV clades CEN.S or EST.S in two replicate sampling transects. Focusing the analysis on 199 voles with evidence of genomic admixture at the individual level (0.1-0.9) supported statistical significance for all 30 loci. Host genomic variation at these SNPs explained up to 37.6% of clade-specific TULV infections. Genes in the vicinity of associated SNPs include SAHH, ITCH and two members of the Syngr gene family, which are involved in functions related to immune response or membrane transport. This study demonstrates the relevance of natural hybrid zones as systems not only for studying processes of evolutionary divergence and speciation, but also for the detection of evolving genetic barriers for specialized parasites., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Hantaviruses are zoonotic pathogens that can cause subclinical to lethal infections in humans. In Europe, five orthohantaviruses are present in rodents: Myodes-associated Puumala orthohantavirus (PUUV), Microtus-associated Tula orthohantavirus, Traemmersee hantavirus (TRAV)/ Tatenale hantavirus (TATV)/ Kielder hantavirus, rat-borne Seoul orthohantavirus, and Apodemus-associated Dobrava-Belgrade orthohantavirus (DOBV). Human PUUV and DOBV infections were detected previously in Lithuania, but the presence of Microtus-associated hantaviruses is not known. For this study we screened 234 Microtus voles, including root voles (Microtus oeconomus), field voles (Microtus agrestis) and common voles (Microtus arvalis) from Lithuania for hantavirus infections. This initial screening was based on reverse transcription-polymerase chain reaction (RT-PCR) targeting the S segment and serological analysis. A novel hantavirus was detected in eight of 79 root voles tentatively named "Rusne virus" according to the capture location and complete genome sequences were determined. In the coding regions of all three genome segments, Rusne virus showed high sequence similarity to TRAV and TATV and clustered with Kielder hantavirus in phylogenetic analyses of partial S and L segment sequences. Pairwise evolutionary distance analysis confirmed Rusne virus as a strain of the species TRAV/TATV. Moreover, we synthesized the entire nucleocapsid (N) protein of Rusne virus in Saccharomyces cerevisiae. We observed cross-reactivity of antibodies raised against other hantaviruses, including PUUV, with this new N protein. ELISA investigation of all 234 voles detected Rusne virus-reactive antibodies exclusively in four of 79 root voles, all being also RNA positive, but not in any other vole species. In conclusion, the detection of Rusne virus RNA in multiple root voles at the same trapping site during three years and its absence in sympatric field voles suggests root voles as the reservoir host of this novel virus. Future investigations should evaluate host association of TRAV, TATV, Kielder virus and the novel Rusne virus and their evolutionary relationships., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

European orthohantaviruses (Puumala orthohantavirus (PUUV); Dobrava-Belgrade orthohantavirus (DOBV), genotype Kurkino; Tula orthohantavirus (TULV)), and Leptospira spp. are small mammal-associated zoonotic pathogens that cause diseases with potentially similar symptoms in humans. We investigated the frequency of Leptospira spp. and hantavirus single and double infections in small mammals from 22 sites in Thuringia, central Germany, during 2017. TULV infections were detected at 18 of 22 sites (mean prevalence 13.8%, 93/674). PUUV infections were detected at four of 22 sites (mean prevalence 1.5%, 7/471), and respective PUUV sequences formed a novel phylogenetic clade, but DOBV infections were not detected at all. Leptospira infections were detected at 21 of 22 sites with the highest overall prevalence in field voles (Microtus agrestis) with 54.5% (6/11) and common voles (Microtus arvalis) with 30.3% (205/676). Leptospira-hantavirus coinfections were found in 6.6% (44/671) of common voles but only in two of 395 bank voles. TULV and Leptospira coinfection probability in common voles was driven by individual (age) and population-level factors. Coinfections seemed to be particularly associated with sites where Leptospira spp. prevalence exceeded 35%. Future investigations should evaluate public health consequences of this strong spatial clustering of coinfections.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

The diversity of viruses probably exceeds biodiversity of eukaryotes, but little is known about the origin and emergence of novel virus species. Experimentation and disease outbreak investigations have allowed the characterization of rapid molecular virus adaptation. However, the processes leading to the establishment of functionally distinct virus taxa in nature remain obscure. Here, we demonstrate that incipient speciation in a natural host species has generated distinct ecological niches leading to adaptive isolation in an RNA virus. We found a very strong association between the distributions of two major phylogenetic clades in Tula orthohantavirus (TULV) and the rodent host lineages in a natural hybrid zone of the European common vole (Microtus arvalis). The spatial transition between the virus clades in replicated geographic clines is at least eight times narrower than between the hybridizing host lineages. This suggests a strong barrier for effective virus transmission despite frequent dispersal and gene flow among local host populations, and translates to a complete turnover of the adaptive background of TULV within a few hundred meters in the open, unobstructed landscape. Genetic differences between TULV clades are homogenously distributed in the genomes and mostly synonymous (93.1%), except for a cluster of nonsynonymous changes in the 5' region of the viral envelope glycoprotein gene, potentially involved in host-driven isolation. Evolutionary relationships between TULV clades indicate an emergence of these viruses through rapid differential adaptation to the previously diverged host lineages that resulted in levels of ecological isolation exceeding the progress of speciation in their vertebrate hosts., Competing Interests: The authors have declared that no competing interests exist.

Human hantavirus disease cases, caused by Puumala virus (PUUV), are mainly recorded in western and southern areas of Germany. This bank vole reservoir survey confirmed PUUV presence in these regions but its absence in northern and eastern regions. PUUV occurrence is associated with the presence of the Western bank vole phylogroup.

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

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.