Puumala hantavirus infection alters the odour attractiveness of its reservoir host

The random-mixing assumptions of many parasite-transmission models are challenged if healthy individuals can alter their behaviour to reduce their risk of infection. Some pathogens reduce the attractiveness of their hosts’ excretions, for example, potentially altering contact rates and thus the predicted force of infection for pathogens transmissible by contact with excretions. For bank voles (Myodes glareolus), contact with contaminated urine is an important route of transmission for Puumala hantavirus (PUUV); however, it is not known whether PUUV infection changes the voles’ urinary odours or their attractiveness. Here, we use a Y-maze to test whether PUUV infection alters the attractiveness of male bank voles’ urine. We presented wild-caught PUUV-free male and female bank voles with PUUV-infected conspecific urine, uninfected urine and a water control, and measured the relative and absolute latency to first visit, number of visits, and total time bank voles spent investigating each treatment over 30 min. PUUV infection significantly altered the bank voles’ initial response to conspecific urine, with fewer visits and less time spent close to infected urine relative to uninfected urine, and less total time spent near the infected urine than the uninfected urine or control. These strong preferences weakened over the 30-min trial, however, partly due to a general decline in male activity, and there were no absolute differences between the treatments overall. This suggests that PUUV infection does change the attractiveness of bank vole urine to conspecifics, and we discuss the implications of these results for random-mixing assumptions.     

Maternal antibodies contribute to sex-based difference in hantavirus transmission dynamics

Individuals often differ in their ability to transmit disease and identifying key individuals for transmission is a major issue in epidemiology. Male hosts are often thought to be more important than females for parasite transmission and persistence. However, the role of infectious females, particularly the transient immunity provided to offspring through maternal antibodies (MatAbs), has been neglected in discussions about sex-biased infection transmission. We examined the effect of host sex upon infection dynamics of zoonotic Puumala hantavirus (PUUV) in semi-natural, experimental populations of bank vole (Myodes glareolus). Populations were founded with either females or males that were infected with PUUV, whereas the other sex was immunized against PUUV infection. The likelihood of the next generation being infected was lower when the infected founders were females, underlying the putative importance of adult males in PUUV transmission and persistence in host populations. However, we show that this effect probably results from transient immunity that infected females provide to their offspring, rather than any sex-biased transmission efficiency per se. Our study proposes a potential contrasting nature of female and male hosts in the transmission dynamics of hantaviruses.     

Impact of Puumala virus infection on maturation and survival in bank voles: a capture-mark-recapture analysis

Many zoonotic diseases are caused by rodent-borne viruses. Major fluctuations in the transmission of these viruses have been related to large changes in reservoir host population numbers due to external factors. However, the impact of the pathogen itself on the demography of its reservoir host is often overlooked. We investigated the impact of Puumala virus (PUUV) on survival and reproductive maturation probability of its reservoir host, the bank vole (Myodes glareolus). Three years (2004-06) of data from nine independent sites in southern Belgium were collected and analyzed with a capture-mark-recapture (CMR) method that includes statistical correction for the variation in capture probability of voles. A multistate model based on four states of reproductive activity and PUUV immunoglobulin G (IgG) antibody status was used to estimate survival and probability of transition from one reproductive or infection state to another. Although survival estimates for reproductively active voles were similar between infected and noninfected individuals, PUUV infection in reproductively inactive voles decreased mean monthly survival by 14%. PUUV infection was associated with a threefold increase in the probability of reproductive maturation in bank voles. Moreover, the probability of PUUV IgG seroconversion was three times higher for reproductively active voles compared to reproductively inactive voles. Our model indicates that PUUV infection may alter bank vole population dynamics by affecting both survival and maturation in its host. Additional studies, using CMR methodology with shorter time intervals between trapping sessions and possibly a longer duration, are needed to confirm these findings.     

Environmental change and disease dynamics: effects of intensive forest management on Puumala hantavirus infection in boreal bank vole populations

Intensive management of Fennoscandian forests has led to a mosaic of woodlands in different stages of maturity. The main rodent host of the zoonotic Puumala hantavirus (PUUV) is the bank vole (Myodes glareolus), a species that can be found in all woodlands and especially mature forests. We investigated the influence of forest age structure on PUUV infection dynamics in bank voles. Over four years, we trapped small mammals twice a year in a forest network of different succession stages in Northern Finland. Our study sites represented four forest age classes from young (4 to 30 years) to mature (over 100 years) forests. We show that PUUV-infected bank voles occurred commonly in all forest age classes, but peaked in mature forests. The probability of an individual bank vole to be PUUV infected was positively related to concurrent host population density. However, when population density was controlled for, a relatively higher infection rate was observed in voles trapped in younger forests. Furthermore, we found evidence of a "dilution effect" in that the infection probability was negatively associated with the simultaneous density of other small mammals during the breeding season. Our results suggest that younger forests created by intensive management can reduce hantaviral load in the environment, but PUUV is common in woodlands of all ages. As such, the Fennoscandian forest landscape represents a significant reservoir and source of hantaviral infection in humans.     

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.     

Hantavirus infections in fluctuating host populations: the role of maternal antibodies

Infected females may transfer maternal antibodies (MatAbs) to their offspring, which may then be transiently protected against infections the mother has encountered. However, the role of maternal protection in infectious disease dynamics in wildlife has largely been neglected. Here, we investigate the effects of Puumala hantavirus (PUUV)-specific MatAbs on PUUV dynamics, using 7 years'' data from a cyclic bank vole population in Finland. For the first time to our knowledge, we partition seropositivity data from a natural population into separate dynamic patterns for MatAbs and infection. The likelihood of young of the year carrying PUUV-specific MatAbs during the breeding season correlated positively with infection prevalence in the overwintered parent population in the preceding spring. The probability of PUUV infection varied between seasons (highest in spring, lowest in late summer) and depended on population structure, but was also, in late autumn, notably, negatively related to summer MatAb prevalence, as well as to infection prevalence earlier in the breeding season. Hence, our results suggest that high infection prevalence in the early breeding season leads to a high proportion of transiently immune young individuals, which causes delays in transmission. This suggests, in turn, that MatAb protection has the potential to affect infection dynamics in natural populations.     

Landscape features and helminth co-infection shape bank vole immunoheterogeneity,with consequences for Puumala virus epidemiology

Heterogeneity in environmental conditions helps to maintain genetic and phenotypic diversity in ecosystems. As such, it may explain why the capacity of animals to mount immune responses is highly variable. The quality of habitat patches, in terms of resources, parasitism, predation and habitat fragmentation may, for example, trigger trade-offs ultimately affecting the investment of individuals in various immunological pathways. We described spatial immunoheterogeneity in bank vole populations with respect to landscape features and co-infection. We focused on the consequences of this heterogeneity for the risk of Puumala hantavirus (PUUV) infection. We assessed the expression of the Tnf-α and Mx2 genes and demonstrated a negative correlation between PUUV load and the expression of these immune genes in bank voles. Habitat heterogeneity was partly associated with differences in the expression of these genes. Levels of Mx2 were lower in large forests than in fragmented forests, possibly due to differences in parasite communities. We previously highlighted the positive association between infection with Heligmosomum mixtum and infection with PUUV. We found that Tnf-α was more strongly expressed in voles infected with PUUV than in uninfected voles or in voles co-infected with the nematode H. mixtum and PUUV. H. mixtum may limit the capacity of the vole to develop proinflammatory responses. This effect may increase the risk of PUUV infection and replication in host cells. Overall, our results suggest that close interactions between landscape features, co-infection and immune gene expression may shape PUUV epidemiology.     

A model system for in vitro studies of bank vole borne viruses

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2. In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.     

The dynamics of murid gammaherpesvirus 4 within wild, sympatric populations of bank voles and wood mice

Murid gammaherpesvirus 4 (MuHV-4) is widely used as a small animal model for understanding gammaherpesvirus infections in man. However, there have been no epidemiological studies of the virus in wild populations of small mammals. As MuHV-4 both infects cells associated with the respiratory and immune systems and attempts to evade immune control via various molecular mechanisms, infection may reduce immunocompetence with potentially serious fitness consequences for individuals. Here we report a longitudinal study of antibody to MuHV-4 in a mixed assemblage of bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) in the UK. The study was conducted between April 2001 and March 2004. Seroprevalence was higher in wood mice than bank voles, supporting earlier work that suggested wood mice were the major host even though the virus was originally isolated from a bank vole. Analyses of both the probability of having antibodies and the probability of initial seroconversion indicated no clear seasonal pattern or relationship with host density. Instead, infection risk was most closely associated with individual characteristics, with heavier males having the highest risk. This may reflect individual variation in susceptibility, potentially related to variability in the ability to mount an effective immune response.     

Inference of cowpox virus transmission rates between wild rodent host classes using space-time interaction

There have been virtually no studies of 'who acquires infection from whom' in wildlife populations, but patterns of transmission within and between different classes of host are likely to be reflected in the spatiotemporal distribution of infection among those host classes. Here, we use a modified form of K-function analysis to test for space-time interaction among bank voles and wood mice infectious with cowpox virus. There was no evidence for transmission between the two host species, supporting previous evidence that they act as separate reservoirs for cowpox. Among wood mice, results suggested that transmission took place primarily between individuals of the opposite sex, raising the possibility that cowpox is sexually transmitted in this species. Results for bank voles indicated that infected females might be a more important source of infection to either sex than are males. The suggestion of different modes of transmission in the two species is itself consistent with the apparent absence of transmission between species.     

Landscape genetics highlights the role of bank vole metapopulation dynamics in the epidemiology of Puumala hantavirus

Rodent host dynamics and dispersal are thought to be critical for hantavirus epidemiology as they determine pathogen persistence and transmission within and between host populations. We used landscape genetics to investigate how the population dynamics of the bank vole Myodes glareolus, the host of Puumala hantavirus (PUUV), vary with forest fragmentation and influence PUUV epidemiology. We sampled vole populations within the Ardennes, a French PUUV endemic area. We inferred demographic features such as population size, isolation and migration with regard to landscape configuration. We next analysed the influence of M. glareolus population dynamics on PUUV spatial distribution. Our results revealed that the global metapopulation dynamics of bank voles were strongly shaped by landscape features, including suitable patch size and connectivity. Large effective size in forest might therefore contribute to the higher observed levels of PUUV prevalence. By contrast, populations from hedge networks highly suffered from genetic drift and appeared strongly isolated from all other populations. This might result in high probabilities of local extinction for both M. glareolus and PUUV. Besides, we detected signatures of asymmetric bank vole migration from forests to hedges. These movements were likely to sustain PUUV in fragmented landscapes. In conclusion, our study provided arguments in favour of source‐sink dynamics shaping PUUV persistence and spread in heterogeneous, Western European temperate landscapes. It illustrated the potential contribution of landscape genetics to the understanding of the epidemiological processes occurring at this local scale.     

Cross-protection against challenge with Puumala virus after immunization with nucleocapsid proteins from different hantaviruses

Hantaviruses are rodent-borne agents that cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome in humans. The nucleocapsid protein (N) is relatively conserved among hantaviruses and highly immunogenic in both laboratory animals and humans, and it has been shown to induce efficient protective immunity in animal models. To investigate the ability of recombinant N (rN) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV and TOPV rN were completely protected. In the group immunized with DOBV rN, 7 of 10 animals were protected, while only 3 of 8 animals were protected in the group immunized with ANDV rN, which is more closely related to PUUV rN than DOBV rN. Humoral and cellular immune responses after rN immunization were also investigated. The highest cross-reactive humoral responses against PUUV antigen were detected in sera from ANDV rN-immunized animals, followed by those from TOPV rN-immunized animals, and only very low antibody cross-reactivity was observed in sera from DOBV rN-immunized animals. In proliferation assays, T lymphocytes from animals immunized with all heterologous rNs were as efficiently recalled in vitro by PUUV rN as were T lymphocytes from animals immunized with homologous protein. In summary, this study has shown that hantavirus N can elicit cross-protective immune responses against PUUV, and the results suggest a more important role for the cellular arm of the immune response than for the humoral arm in cross-protection elicited by rN.     

Predation rate, prey preference and predator switching: experiments on voles and weasels

We studied the predation rate and prey selection of the least weasel ( Mustela nivalis nivalis ) on its two most common prey species in boreal environments, the bank vole ( Clethrionomys glareolus ) and the field vole ( Microtus agrestis ), in large outdoor enclosures. We also studied the response of weasels to odours of the two species in the laboratory. The enclosure experiment was conducted using constant vole densities (16 voles/ha) but with varying relative abundance of the two species. Weasels showed higher predation rates on bank voles, and males had higher predation rate than females. Females killed disproportionately more of the more abundant prey species, but they preferred bank voles to field voles when both were equally available. Overall, the predation rate also increased with increasing abundance of bank voles. Therefore our results are in agreement with earlier laboratory results showing preference for bank voles, even if no intrinsic preference for odours of either species was observed in our laboratory study. We suggest that the least weasel hunts according to prey availability, prey aggregation and suitability of hunting habitat, and that this causes the observed dependence of least weasels on field voles and emphasises the role of the field vole in the vole-weasel interaction in cyclic vole populations. Furthermore, our results suggest that predation by weasels may facilitate the coexistence of the two vole species via predator switching, and that it may cause the observed synchrony in dynamics between vole species.     

Gene conversion in the mitochondrial genome on interspecific hybridization in voles of the <Emphasis Type="Italic">Clethrionomys</Emphasis> genus

The phenomenon of interspecific hybridization accompanied by transfer of the mitochondrial genome from the northern red-backed vole (Clethrionomys rutilus) to the bank vole (Cl. glareolus) in northeastern Europe is well known already for 25 years. However, the possibility of recombination between homologous segments of maternal and paternal mtDNAs of the voles during fertilization was not previously studied. Analysis of data on variability of nucleotide sequences of the mitochondrial gene for cytochrome b in populations of red-backed and bank voles in the area of their sympatry has shown that as a result of interspecific hybridization, the mitochondrial gene pool of bank voles contains not only mtDNA haplotypes of red-backed vole females, but also mtDNA haplotypes of bank voles bearing short nucleotide tracts of red-backed vole mtDNA. This finding supports the hypothesis that an incomplete elimination of red-backed vole paternal mtDNA during the interspecific hybridization between bank vole females and red-backed vole males leads to the gene conversion of bank vole maternal mtDNA tracts by homologous ones of mtDNA of red-backed vole males.     

Direct interference or indirect exploitation? An experimental study of fitness costs of interspecific competition in voles

Studies on competing mammalian species in the past have focused mainly on the competitive exclusion of one species from the preferred habitat of the other. Investigations on effects of competition and coexistence on individual fitness are rare . In this study we were able to measure effects of interspecific competition on major fitness components, using a system with two vole species in asymmetric competition. Survival, reproduction and space use of bank vole Clethrionomys glareolus females were monitored in 32 enclosed populations over four replicates of eight parallel run enclosures. Into half of the enclosures we introduced an additional number of field voles Microtus agrestis , a dominant competitor.
Survival of bank vole females was lower under competitive conditions. Total number of breeding females was lower in populations coexisting with competitors. Territory size of bank vole females decreased. Females body weight and litter size bank vole litters conceived during the experiment were not affected by interspecific competition. These characteristics should respond to differences in food resources, and territory size should increase if food was scarce, thus we found no indication of direct exploitation competition between the two species. Space use was overlapping between the species, but individuals of both species were never caught together in the same trap, indicating avoidance behaviour.
We conclude that adult bank vole females do suffer fitness consequences through interference competition with field voles, probably basing on increased number of aggressive encounters in the presence of the dominant species. Our results suggest, that direct interference rather than indirect exploitation competition may be the cause for observed fitness decrease in bank vole females.     

Female-biased infection and transmission of the gastrointestinal nematode Trichuris arvicolae infecting the common vole, Microtus arvalis

Previous studies addressing the importance of host gender in parasite transmission have shed light on males as the more important hosts, with the higher transmission potential of males being explained by the fact that they often harbour higher parasite loads than females. However, in some systems females are more heavily infected than males and may be responsible for driving infection under such circumstances. Using a wild population of common voles (Microtus arvalis), we showed that females were more frequently infected by the intestinal nematode Trichuris arvicolae than males (i.e. prevalence based on the presence of eggs in the faeces) and that females were shedding greater numbers of parasite eggs per gram of faeces (EPG) than males. By applying an anthelmintic treatment to either male or female voles, we demonstrated that treating females significantly reduced parasite burdens (i.e. prevalence and EPG) of both male and female hosts, while treating males only reduced parasite burden in males. These findings indicate that in this female-biased infection system females play a more important role than males in driving the dynamics of parasite transmission.     

Introduced Siberian chipmunks are more heavily infested by ixodid ticks than are native bank voles in a suburban forest in France

By serving as hosts for native vectors, introduced species can surpass native hosts in their role as major reservoirs of local pathogens. During a 4-year longitudinal study, we investigated factors that affected infestation by ixodid ticks on both introduced Siberian chipmunks Tamias sibiricus barberi and native bank voles Myodes glareolus in a suburban forest (Forêt de Sénart, Ile-de-France). Ticks were counted on adult bank voles and on adult and young chipmunks using regular monthly trapping sessions, and questing ticks were quantified by dragging. At the summer peak of questing Ixodes ricinus availability, the average tick load was 27-69 times greater on adult chipmunks than on adult voles, while average biomass per hectare of chipmunks and voles were similar. In adult chipmunks, individual effects significantly explained 31% and 24% of the total variance of tick larvae and nymph burdens, respectively. Male adult chipmunks harboured significantly more larvae and nymphs than adult females, and than juveniles born in spring and in summer. The higher tick loads, and more specifically the ratio of nymphs over larvae, observed in chipmunks may be caused by a higher predisposition - both in terms of susceptibility and exposure - to questing ticks. Tick burdens were also related to habitat and seasonal variation in age- and sex-related space use by both rodents. Introduced chipmunks may thus have an important role in the dynamics of local vector-borne pathogens compared with native reservoir hosts such as bank voles.     

Maternal investment in relation to sex ratio and offspring number in a small mammal – a case for Trivers and Willard theory?

1.  Optimal parental sex allocation depends on the balance between the costs of investing into sons vs. daughters and the benefits calculated as fitness returns. The outcome of this equation varies with the life history of the species, as well as the state of the individual and the quality of the environment.
2.  We studied maternal allocation and subsequent fecundity costs of bank voles, Myodes glareolus , by manipulating both the postnatal sex ratio (all-male/all-female litters) and the quality of rearing environment (through manipulation of litter size by −2/+2 pups) of their offspring in a laboratory setting.
3.  We found that mothers clearly biased their allocation to female rather than male offspring regardless of their own body condition. Male pups had a significantly lower growth rate than female pups, so that at weaning, males from enlarged litters were the smallest. Mothers produced more milk for female litters and also defended them more intensively than male offspring.
4.  The results agree with the predictions based on the bank vole life history: there will be selection for greater investment in daughters rather than sons, as a larger size seems to be more influencial for female reproductive success in this species. Our finding could be a general rule in highly polygynous, but weakly dimorphic small mammals where females are territorial.
5.  The results disagree with the narrow sense Trivers & Willard hypothesis, which states that in polygynous mammals that show higher variation in male than in female reproductive success, high-quality mothers are expected to invest more in sons than in daughters.     

Serological survey for viral pathogens in Turkish rodents

Wild rodents (n = 330) were trapped around the villages of Altindere and Co?andere (Ma?ka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihem?in, Rize Province), and Bozdag (Odemi?, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.