Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

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.

Endemic Hantavirus in Field Voles, Northern England.

We report a PCR survey of hantavirus infection in an extensive field vole (Microtus agrestis) population present in the Kielder Forest, northern England. A Tatenale virus-like lineage was frequently detected (≈17% prevalence) in liver tissue. Lineages genetically similar to Tatenale virus are likely to be endemic in northern England.

Mycobacterium microti tuberculosis in its maintenance host, the field vole (Microtus agrestis): characterization of the disease and possible routes of transmission.

The field vole (Microtus agrestis) is a known maintenance host of Mycobacterium microti. Previous studies have shown that infected animals develop tuberculosis. However, the disease is also known in cats and is sporadically reported from humans and other mammalian species. We examined trapped field voles from an endemic area, using a range of diagnostic approaches. These confirmed that a combination of gross and histological examination with culture is most appropriate to identify the true prevalence of the disease, which was shown to be more than 13% at times when older animals that have previously been shown to be more likely to develop the disease dominate the population. The thorough pathological examination of diseased animals showed that voles generally develop systemic disease with most frequent involvement of spleen and liver, followed by skin, lymph nodes, and lungs. The morphology of the lesions was consistent with active disease, and their distribution suggested skin wounds or oral and/or aerogenic infection as the main portal of entry. The demonstration of mycobacteria in open skin lesions, airways, and salivary glands indicated bacterial shedding from the skin and with sputum and saliva. This suggests not only the environment but also direct contact and devouring as likely sources of infection.

Spatial and Temporal Dynamics and Molecular Evolution of Tula orthohantavirus in German Vole Populations.

Tula orthohantavirus (TULV) is a rodent-borne hantavirus with broad geographical distribution in Europe. Its major reservoir is the common vole (Microtus arvalis), but TULV has also been detected in closely related vole species. Given the large distributional range and high amplitude population dynamics of common voles, this host-pathogen complex presents an ideal system to study the complex mechanisms of pathogen transmission in a wild rodent reservoir. We investigated the dynamics of TULV prevalence and the subsequent potential effects on the molecular evolution of TULV in common voles of the Central evolutionary lineage. Rodents were trapped for three years in four regions of Germany and samples were analyzed for the presence of TULV-reactive antibodies and TULV RNA with subsequent sequence determination. The results show that individual (sex) and population-level factors (abundance) of hosts were significant predictors of local TULV dynamics. At the large geographic scale, different phylogenetic TULV clades and an overall isolation-by-distance pattern in virus sequences were detected, while at the small scale (<4 km) this depended on the study area. In combination with an overall delayed density dependence, our results highlight that frequent, localized bottleneck events for the common vole and TULV do occur and can be offset by local recolonization dynamics.

Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports Its Classification as a Novel Species.

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus.

Detection of Francisella tularensis in three vole species in Central Europe.

Francisella tularensis is a zoonotic, gram-negative bacterium that causes tularemia in humans. Depending on its subspecies and the route of transmission, mild to lethal courses have been reported. F. tularensis subsp. holarctica is the only subspecies found in Europe and affects a plenitude of vertebrates including lagomorphs and rodents. Population outbreaks of certain rodent species are likely to be involved in the transmission of this pathogen. This molecular survey aims to evaluate the presence of F. tularensis in small mammals from three Central European countries. Using a real-time polymerase chain reaction, F. tularensis DNA was detected in common voles (Microtus arvalis) from Switzerland and in field voles (Microtus agrestis) and a bank vole (Myodes glareolus) from Germany, but not in any other small mammal species. All common voles from the Czech Republic were negative for F. tularensis DNA. The prevalence in the three vole species varied between 1.3% and 3.0%. In conclusion, Francisella tularensis DNA was detected in three vole species in two of three countries investigated. The observed low prevalence raises questions on the role of voles for the transmission of Francisella tularensis in Central Europe.

Characterization of the complete mitochondrial genome of short-tailed field vole, Microtus agrestis.

In this study, the complete mitochondrial genome of short-tailed field vole, Microtus agrestis, was determined through sequencing of PCR fragments. The complete mitochondrial genome of M. agrestis was 16,538 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 15.7% A, 27.5% T, 25.5% C, and 31.4% G, with a total G + C content of 56.9%. By phylogenetic analysis using Bayesian method, M. agrestisa showed the closest relationship with the southern vole (Microtus rossiaemeridionalis).

Dampening of population cycles in voles affects small mammal community structure, decreases diversity, and increases prevalence of a zoonotic disease.

Long-term decline and depression of density in cyclic small rodents is a recent widespread phenomenon. These observed changes at the population level might have cascading effects at the ecosystem level. Here, we assessed relationships between changing boreal landscapes and biodiversity changes of small mammal communities. We also inferred potential effects of observed community changes for increased transmission risk of Puumala virus (PUUV) spread, causing the zoonotic disease nephropatica epidemica in humans. Analyses were based on long-term (1971-2013) monitoring data of shrews and voles representing 58 time series in northern Sweden. We calculated richness, diversity, and evenness at alpha, beta, and gamma level, partitioned beta diversity into turnover (species replacement) and nestedness (species addition/removal), used similarity percentages (SIMPER) analysis to assess community structure, and calculated the cumulated number of PUUV-infected bank voles and average PUUV prevalence (percentage of infected bank voles) per vole cycle. Alpha, beta, and gamma richness and diversity of voles, but not shrews, showed long-term trends that varied spatially. The observed patterns were associated with an increase in community contribution of bank vole (Myodes glareolus), a decrease of gray-sided vole (M. rufocanus) and field vole (Microtus agrestis) and a hump-shaped variation in contribution of common shrew (Sorex araneus). Long-term biodiversity changes were largely related to changes in forest landscape structure. Number of PUUV-infected bank voles in spring was negatively related to beta and gamma diversity, and positively related to turnover of shrews (replaced by voles) and to community contribution of bank voles. The latter was also positively related to average PUUV prevalence in spring. We showed that long-term changes in the boreal landscape contributed to explain the decrease in biodiversity and the change in structure of small mammal communities. In addition, our results suggest decrease in small mammal diversity to have knock-on effects on dynamics of infectious diseases among small mammals with potential implications for disease transmission to humans.

Establishment of superovulation procedure in Japanese field vole, Microtus montebelli.

Japanese field vole (Microtus montebelli) is a wild-derived rodent and have unique characteristic. Thus, these species have been expected as model animal. This study was performed to develop novel superovulation procedure for Japanese field vole. First, when 30 IU pregnant mare's serum gonadotropin (PMSG) and 30 IU human chorionic gonadotropin (hCG) were administrated 48 hours apart, females showed higher response to hCG compared with three concentrations of PMSG. Second, to effectively induce ovulation on females after vaginal opening, they were mated with vasectomized male instead of hCG administration. Average number of ovulated oocytes using PMSG mating (13.9 ± 1.9 oocytes) was higher than PMSG-hCG (control; 6.9 ± 2.3 oocytes) or PMSG-hCG mating (6.8 ± 0.8 oocytes). Finally, we attempted superovulation using GnRH agonist (GnRHa). With this treatment, we speculated that GnRHa might induce endogenous luteinizing hormone releasing to cause ovulation. Such superovulation was performed with 30 IU PMSG and different concentration of 20% polyvinylpyrrolidone-GnRHa (15, 30, 45, and 60 µg/kg). As results, average number of ovulated oocytes was highest with 30 µg/kg GnRHa (14.5 ± 4.1 oocytes). The numbers of ovulated oocytes of other concentrations were 5.0 ± 1.4 (15 µg/kg), 12.8 ± 2.7 (45 µg/kg), and 8.8 ± 3.7 oocytes (60 µg/kg). Nuclear status of most collected oocytes was the second meiotic division (range, 94.3%-100%). These superovulation procedures will be useful for development of in vitro culture systems and assisted reproductive technologies for not only Japanese field vole but also other voles.

Spatial analyses of wildlife contact networks.

Datasets from which wildlife contact networks of epidemiological importance can be inferred are becoming increasingly common. A largely unexplored facet of these data is finding evidence of spatial constraints on who has contact with whom, despite theoretical epidemiologists having long realized spatial constraints can play a critical role in infectious disease dynamics. A graph dissimilarity measure is proposed to quantify how close an observed contact network is to being purely spatial whereby its edges are completely determined by the spatial arrangement of its nodes. Statistical techniques are also used to fit a series of mechanistic models for contact rates between individuals to the binary edge data representing presence or absence of observed contact. These are the basis for a second measure that quantifies the extent to which contacts are being mediated by distance. We apply these methods to a set of 128 contact networks of field voles (Microtus agrestis) inferred from mark-recapture data collected over 7 years and from four sites. Large fluctuations in vole abundance allow us to demonstrate that the networks become increasingly similar to spatial proximity graphs as vole density increases. The average number of contacts, 〈k〉, was (i) positively correlated with vole density across the range of observed densities and (ii) for two of the four sites a saturating function of density. The implications for pathogen persistence in wildlife may be that persistence is relatively unaffected by fluctuations in host density because at low density 〈k〉 is low but hosts move more freely, and at high density 〈k〉 is high but transmission is hampered by local build-up of infected or recovered animals.

MITOCHONDRIAL DNA VARIATION IN THE FIELD VOLE (MICROTUS AGRESTIS): REGIONAL POPULATION STRUCTURE AND COLONIZATION HISTORY.

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) was used to examine the genetic structure among field voles (Microtus agrestis) from southern and central Sweden. A total of 57 haplotypes was identified in 158 voles from 60 localities. Overall mtDNA diversity was high, but both haplotype and nucleotide diversity exhibited pronounced geographic heterogeneity. Phylogenetic analyses revealed a shallow tree with seven primary mtDNA lineages separated by sequence divergences ranging from 0.6% to 1.0%. The geographic structure of mtDNA diversity and lineage distribution was complex but strongly structured and deviated significantly from an equilibrium situation. The extensive mtDNA diversity observed and the recent biogeographic history of the region suggests that the shallow mtDNA structure in the field vole cannot be explained solely by stochastic lineage sorting in situ or isolation by distance. Instead, the data suggest that the genetic imprints of historical demographic conditions and vicariant geographic events have been preserved and to a large extent determine the contemporary geographic distribution of mtDNA variation. A plausible historical scenario involves differentiation of mtDNA lineages in local populations in glacial refugia, a moving postglacial population structure, and bottlenecks and expansions of mtDNA lineages during the postglacial recolonization of Sweden. By combining the mtDNA data with an analysis of Y-chromosome variation, a specific population unit was identified in southwestern Sweden. This population, defined by a unique mtDNA lineage and fixation of a Y-chromosome variant, probably originated in a population bottleneck in southern Sweden about 12,000 to 13,000 calendar years ago.

A CONTACT ZONE WITH NONCOINCIDENT CLINES FOR SEX-SPECIFIC MARKERS IN THE FIELD VOLE (MICROTUS AGRESTIS).

A field vole (Microtus agrestis) population characterized by unique mitochondrial DNA (mtDNA) and Y chromosome markers occurs in southwest Sweden. A contact zone between this Lund (Lu) population and a standard (St) population was examined with two sex-specific genetic markers. The field vole mtDNA and Y chromosome clines display a remarkable lack of coincidence, rarely observed in contact zones. The cline width for both markers is about 50 km, but the two clines are displaced from each other: the mtDNA cline is found in the central part of the study area, whereas the cline for the Y chromosome is located in the eastern part. Thus, the absolute width of the Lu-St zone spans about 95-110 km. As a result of the cline shift, all male hybrids carry the Lu-Y chromosome and St-type mtDNA. The other possible male hybrid class is lacking. The distinct noncoincidence of the mtDNA and Y chromosome clines is most likely explained by selection against male hybrids with the St-Y chromosome and Lu-mtDNA. It is possible that incompatibilities between the maternal genome of Lu-type animals and the paternal genome of St-type individuals exist. However, alternative explanations based on neutrality or selective advantage cannot be totally dismissed.