Effect of temperature on antioxidant defense and innate immunity in Brandt’s voles

Ambient temperature is an importa nt factor in flue ncing many physiological processes, in eluding antioxidant defense and immunity. In the present study, we tested the hypothesis that antioxidant defense and immunity are suppressed by high and low temperature treatme nt in Bran dt's voles (Lasiopodomys brandtii). Thirty male voles were randomly assigned into different temperature groups (4, 23, and 32℃, n=10 for each group), with the treatment course lasting for 27 d. Results showed that low temperature in creased gross en ergy in take (GEI) and liver, heart, and kidney mass, but decreased body fat mass and dry carcass mass. With the decline in temperature, hydrogen peroxide (H2O2) concentration, which is indicative of reactive oxyge n species (ROS) levels, in creased in the liver, decreased in the heart, and was unchanged in the kidney, testis, and small intestine. Lipid peroxidation indicated by malonaldehyde (MDA) content in the liver, heart, kidney, testis, and small intestine did not differ among groups, implying that high and low temperature did not cause oxidative damage. Similarly, superoxide dismutase (SOD) and catalase (CAT) activities and total antioxidant capacity (TAOC) in the five tissues did not respond to low or high temperature, except for elevation of CAT activity in the testis upon cold exposure. Bacteria killing capacity, which is indicative of innate immunity, was nearly suppressed in the 4℃ group in contrast to the 23℃ group, whereas spleen mass and white blood cells were un affected by temperature treatment. The levels of testosterone, but not corticostero ne, were in flue need by temperature treatment, though neither were correlated with innate immunity, H2O2 and MDA levels, or SOD, CAT, and TAOC activity in any detected tissues. Overall, these results showed that temperature had different in flue nces on oxidative stress, an tioxida nt en zymes, and immunity, which depended on the tissues and parameters tested. Up-regulation or maintenance of an tioxida nt defe nse might be an importa nt mechanism for voles to survive highly variable environmental temperatures.

Spontaneous emergence of overgrown molar teeth in a colony of Prairie voles(Microtus ochrogaster)

Continuously growing incisors are common to all rodents, which include the Microtus genus of voles. However, unlike many rodents, voles also possess continuously growing molars. Here, we report spontaneous molar defects in a population of Prairie voles （Microtus ochrogaster）. We identified bilateral protuberances on the ventral surface of the mandible in several voles in our colony. In some cases, the protuberances broke through the cortical bone. The mandibular molars became exposed and infected, and the maxillary molars entered the cranial vault. Visualisation upon soft tissue removal and microcomputed tomography （microCT） analyses confirmed that the protuberances were caused by the overgrowth of the apical ends of the molar teeth. We speculate that the unrestricted growth of the molars was due to the misregulation of the molar dental stem cell niche. Further study of this molar phenotype may yield additional insight into stem cell regulation and the evolution and development of continuously growing teeth.

Mating behavior induces changes of expression of Fos protein,plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB) of the male mandarin vole Microtus mandarinus

In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus,the radioimmunoassay(RIA)and immunohistochemistry methods were used to investigate the differences in plasma testosterone(T)concentrations and distribution of T immunoreactive neurons(T-IRs),androgen receptor immunoreactive neurons(AR-IRs)and Fos protein immunoreactive neurons(Fos-IRs)in the accessory olfactory bulb(AOB)and the main olfactory bulb(MOB)following exposure to clean hard-wood shavings(control group),soiled bedding(exposure group)or contact with an estrous female(mating group).Results showed that plasma T concentration was significantly higher in the mating group than that in the exposure group,and both the mating group and the exposure group displayed significantly higher plasma T concentration than the control group.T-IRs,AR-IRs and Fos-IRs were investigated with the immunohistochemistry method in granule cell(GC)and mitral cell(MC)of the MOB and the AOB in the three groups.There were significantly more T-IRs,AR-IRs and Fos-IRs in MC and GC of the AOB in the mating group than that in the exposure group or the control group.T-IRs,AR-IRs and Fos-IRs did not show significant differences between the exposure group and the control group.Furthermore,obvious differences in MC and GC of the MOB were not found among the three groups.The results confirm that both changes of T and AR in the AOB might be underlying mating behavior in the adult male mandarin voles.

Small mammal community composition impacts bank vole (Clethrionomys glareolus) population dynamics and associated seroprevalence of Puumala orthohantavirus

Rodents are important reservoirs for zoonotic pathogens that cause diseases in humans.Biodiversity is hypoth-esized to be closely related to pathogen prevalence through multiple direct and indirect pathways.For example,the presence of non-host species can reduce contact rates of the main reservoir host and thus reduce the risk of transmission(“dilution effect”).In addition,an overlap in ecological niches between two species could lead to increased interspecific competition,potentially limiting host densities and reducing density-dependent pathogen transmission processes.In this study,we investigated the relative impact of population-level regulation of direct and indirect drivers of the prevalence of Puumala orthohantavirus(PUUV)in bank voles(Clethrionomys glareolus)during years with high abundance.We compiled data on small mammal community composition from four regions in Germany between 2010 and 2013.Structural equation modeling revealed a strong seasonality in PUUV control mechanisms in bank voles.The abundance of shrews tended to have a negative relationship with host abundance,and host abundance positively influenced PUUV seroprevalence,while at the same time increasing the abundance of competing non-hosts like the wood mouse(Apodemus sylvaticus)and the yellow-neckedfield mouse(Apodemusflavicollis)were associated with reduced PUUV seroprevalence in the host.These results indicate that for PUUV in bank voles,dilution is associated with increased interspecific competition.Anthropogenic pressures leading to the decline of Apodemus spp.in a specific habitat could lead to the amplification of mechanisms promoting PUUV transmission within the host populations.

Ecology and social behavior of the social vole Microtus socialis: A generalized review

The present review is a compilation of the published data on the ecology and social behavior of the social vole.Field studies provide evidence that these voles live in family groups consisting of 1 adult male,1 or 2 breeding females,and their offspring(1 or 2 litters).The social vole is capable of year-round reproduction,but in arid regions,the voles demonstrate seasonality in breeding.The mating system of this species may be defined as behavioral monogamy.A typical feature of the space use system is territoriality characterized by a locus-dependent dominance in relationships between neighboring breeding pairs as well as family groups.The family group social organization may be defined as consistent relationships without a dominant hierarchy.Social voles are cooperative in defending their territories,constructing burrows,digging underground tunnels,maintaining nests,and raising young.Cooperation appears to enhance the survival of family groups of this species under the extreme climaticconditionsof Central Asia.

Guild composition and habitat use of voles in 2 forest landscapes in south-eastern Norway

It is widely believed that intensive forestry has influenced small mammal population dynamics,and thereby the entire mammalian community in Fennoscandian boreal forests.The nature of these impacts on the different species is subject to debate.We live-trapped voles between 2006 and 2009 in 2 commercially harvested forests in south-eastern Norway.We investigated the variation in vole abundance among habitat types(e.g.mature forest and clear-cut)and the hypothesis that graminivorous species such as field voles(Microtus agrestis L.)benefit from clear-cuts at the expense of forest dwellers(i.e.the bank vole,Myodes glareolus Schreb.),using fine-scale descriptors of the ground vegetation.We could not find support for the hypothesis that field voles show a preference for clear-cuts,and their overall abundance was low,while bank voles were the dominant species in all habitat types,including clear-cuts in the peak and pre-peak years.We found a positive association between bank vole abundance and bilberry(Vaccinium myrtillus L.)availability rather than a specific habitat type.Low field vole density in clear-cuts might be due to variation in local productivity and ground vegetation as well as to large variation in the species temporal dynamics.The latter is particularly associated with the widespread decline of field voles in Scandinavia.Logging has the potential to negatively affect bank vole population dynamics because of the negative effect on bilberry development.

Relationship between El Nino /South Oscillation(ENSO) and population outbreaks of some lemmings and voles in Europe

Ecologists have been puzzled by population cycles of lemmings and voles for the over 70 years. At present, our understanding and explanation to this phenomenon remain controversial. Recently, El Nino/South Oscillation has attracted attention of ecologists on its links with population outbreaks of terrestrial animals. This paper aims to investigate the statistical relationship between outbreaks of microtine rodents and ENSO events by scanning available literature. During 1862-1894, outbreaks of Norway lemmings in Norway tended to occur in the Southern Oscillation Index (SOI) peak years or 1 year after the SOI peak years with an approximate significance level (p = 0.057). During 1885-1931, outbreaks of voles in France tended to occur 1 year before the SOI peak years (p = 0.01). During 1946-1993, outbreaks of lemmings and voles in North Finland tended to occur 1 year before the SOI peak years with a significant level (p = 0.022); the peaks of population abundance corresponded well to the SOI trough years

Anti-fertility effect of levonorgestrel and quinestrol in Brandt’s voles(Lasiopodomys brandtii)

The combination of levonorgestrel and quinestrol(EP-1)has been shown to have anti-fertility effects on several wild rodents,but the mechanism underlying these effects is poorly understood.We investigated the effects of EP-1 and each of its components,levonorgestrel(P)and quinestrol(E),on the fertility of Brandt’s voles(Lasiopodomys brandtii)by using a gastric gavage method.The doses for EP-1,E and P were 1,0.34 and 0.66 mg/kg body weight,respectively.Male voles(n=98)were treated daily for 5 or 14 days,then the testes and epididymides were collected,weighed and examined histologically at 30(D30),60(D60)or 90(D90)days after the end of treatment.Four males were allowed to mate with normal females at D90.Female voles(n=75)were treated for 3 days and a further 3 days after a 7-day interval.The uteri and ovaries were weighed and examined histologically at 15(D15),30(D30)or 75(D75)days after the end of treatment.Each of three females were mated with fertile males at D30 and D75,respectively.Our results indicated that quinestrol(E)significantly decreased the sperm numbers in the testes as well as the weight of the testes and epididymides,with both of these tissues showing obvious structural abnormalities,and significantly reduced the litter size and the pup weight for females mated with males of the E treatment group.For female voles,treatment with E,P or EP-1 resulted in no marked influence on the fertility status.These data indicate that quinestrol(E)alone has a significant anti-fertility effect on male Brandt’s voles,but is ineffective in combination with levonorgestrel(P).

Effect of ENSO-driven precipitation on population irruptions of the Yangtze vole Microtus fortis calamorum in the Dongting Lake region of China

The Yangtze vole(Microtus fortis Buechner,1889)is a small herbivore species that inhabits lake beaches in the Dongting Lake region along the Yangtze River in Southern China.Its population shows strong oscillations during the wet season due to summer precipitation-induced immigration away from the lake into adjacent rice fields.The effect of El Niño-Southern Oscillation-driven precipitation on population abundance and growth of the vole species is not fully understood.We undertook an analysis of the combined data of 4 time series covering 1981–2006 from 4 different sites and a separate analysis on a single time series(1981–2006)from one site.Our results demonstrate that a dual effect of El Niño-Southern Oscillation-driven precipitation on the population abundance of voles is timedependent:precipitation in the current year has a positive effect,whereas precipitation in the previous year has a negative effect.The dual effect of precipitation on vole population is well explained by the unique interactions among vole population,precipitation water level and the lake beach habitat around Dongting Lake.We found that drier than average weather of the previous year benefited voles because their breeding habitats,lake beaches,were exposed for long stretches of time.Wet weather was found to increase the number of voles inhabiting rice fields because as the water level of the lake rose they were forced from beaches into surrounding rice fields.Summer precipitation in the Dongting Lake region was found to be positively associated with the sea surface temperature(SST)of the eastern tropical Pacific Ocean of the previous year and winter SST and spring SST of the current year.Annual rates of increase in the vole population of the reconstructed time series are negatively associated with the vole abundance and autumn precipitation of the previous year and winter precipitation of the current years.These results suggest that both extrinsic and density-dependent intrinsic factors may affect population dynamics of the Yangtze voles.

Kinship analysis reveals reproductive success skewed toward overwintered Brandt’s voles in semi-natural enclosures

Age structure and seasonality influence the population fluctuations of small rodents.Age determines body weight and social experience,while seasonality regulates the duration of the breeding season and onset of sex­ual maturity in newborn offspring.Therefore,reproductive success and skew usually occur in different age groups.Brandt’s vole(Lasiopodomys brandtii)is a social,short-lived and seasonal breeding small rodent with a dramatic seasonal population fluctuation,but reproductive skew is not fully understood in this species.In the present study,we determined kinship in semi-natural enclosure populations using microsatellite markers based on genotyping,analyzed the reproductive skew between sexes and between overwintered and newborn voles,and monitored variation in male reproductive activity by testing fecal testosterone levels throughout the year.Overwintered voles had the most reproductive success along with a striking increase in the population size in the enclosures,with all biological fathers and 77.8%of biological mothers,which had 100%and 87%of the to­tal offspring,respectively.Compared to overwintered voles,reproductive skews were significantly higher in po­tential overwintered and newborn parents,implying the possible reproductive suppression of newborn voles by dominant overwintered voles.Moreover,both heavier body weight and higher testosterone levels in overwin­tered males supported their potential social status in the population.Our study provided new evidence for repro­ductive skew and differentiation of postnatal gonadal development patterns of different age groups in Brandt’s vole.

Evolutionary history of mountain voles of the subgenus Aschizomys (Cricetidae, Rodentia), inferred from mitochondrial and nuclear markers

In this study,we present an assessment of the evolutionary history and phylogenetic relationships of Asian mountain voles of the subgenus Aschizomys,genus Alticola,based on extensive sampling and phylogenetic analyses of data from mitochondrial and nuclear markers.Two species of this subgenus are widespread in the mountain areas of north-eastern Asia.However,both their distribution and taxonomic borders remained questionable for more than 100 years.Our study showed discordance in the phylogenetic patterns between nuclear and mtDNA markers.We found that mtDNA in A.lemminus is paraphyletic relative to A.macrotis,but nuclear markers demonstrated reciprocal monophyly.According to species distribution modeling,ranges of A.macrotis and A.lemminus experienced a secondary contact during the Last Glacial Maximum(approximately 22 kyr BP),and thus a hybridization event seems plausible during that period.Species tree analyses recovered a sister group relationship between the two species of the Aschizomys subgenus,with an estimated divergence date of around 0.8 Ma.Our results provided good support for currently recognized subspecies within both A.macrotis and A.lemminus based on mitochondrial and nuclear datasets.A new,yet undescribed form,supposedly of a subspecific status within A.lemminus,was found in the Bureinskiy Range in the Khabarovsk area.This finding expands the current species distribution range further to the southeast.

Suggesting the use of integrated methods for vole population management in alfalfa fields

Human-made agro-ecosystems lack negative feedback controls, which may result in rodent population explosions,causing considerable economic loss and thus increasing human–wildlife conflicts. The traditional solution is theextensive use of rodenticides, which have a negative impact on the environment. In Beit-Sheaan Valley, Israel,which is located on a major route for migrating birds, poisoning of voles may result in the death of birds that preyon the dead voles (e.g. storks and herons). In the present essay we discuss some integrated methods, based on theimplementation of ecological principles in agro-ecosystems, that could be used to decrease the use of rodenticides.The first method is light interference during winter (long scotophase), which can result in the death of the socialvole Microtus socialis kept in enclosures under natural conditions. Under laboratory conditions such interferencecauses a decrease in thermoregulatory ability in the cold. As light interference is a source of pollution, only activevole burrows should be illuminated. The second method involves using the mobile irrigation pipelines to flood thevole burrows and force them to escape into the open, where diurnal birds such as storks and herons will prey onthem. The third method involves the use of nesting boxes for barn owls and stands for diurnal raptors, both ofwhich prey on voles. The great advantage of using an integrated approach is that voles cannot become adapted toany one of the methods. Such integrated methods, together with appropriate treatment of alfalfa fields by farmers,can provide an efficient and sustainable pest control approach.

Habitat fragmentation, vole population fluctuations, and the ROMPA hypothesis: An experimental test using model landscapes

Increased habitat fragmentation leads to smaller size of habitat patches and to greater distance between patch­es.The ROMPA hypothesis(ratio of optimal to marginal patch area)uniquely links vole population fluctuations to the composition of the landscape.It states that as ROMPA decreases(fragmentation increases),vole popu­lation fluctuations will increase(including the tendency to display multi-annual cycles in abundance)because decreased proportions of optimal habitat result in greater population declines and longer recovery time after a harsh season.To date,only comparative observations in the field have supported the hypothesis.This paper re­ports the results of the first experimental test.I used prairie voles,Microtus ochrogaster,and mowed grassland to create model landscapes with 3 levels of ROMPA(high with 25%mowed,medium with 50%mowed and low with 75%mowed).As ROMPA decreased,distances between patches of favorable habitat(high cover)in­creased owing to a greater proportion of unfavorable(mowed)habitat.Results from the first year with intensive live trapping indicated that the preconditions for operation of the hypothesis existed(inversely density depen­dent emigration and,as ROMPA decreased,increased per capita mortality and decreased per capita movement between optimal patches).Nevertheless,contrary to the prediction of the hypothesis that populations in land­scapes with high ROMPA should have the lowest variability,5 years of trapping indicated that variability was lowest with medium ROMPA.The design of field experiments may never be perfect,but these results indicate that the ROMPA hypothesis needs further rigorous testing.

Early developmental exposure to inorganic mercury does not alter affiliative behavior of adult prairie voles(Microtus ochrogaster)

Mercury chloride exposure through drinking water in adult male prairie voles(Microtus ochrogaster)has been shown to alter their social behavior.Here,we examined the potential disruption of adult social behavior in prairie voles that were exposed to 60 ppm mercury during early development.We used a cross-fostering approach to test the effects of mercury exposure:(1)from conception until birth;(ii)from birth until weaning;and(iii)from conception until weaning,on adult affiliative behavior.Untreated and mercury-treated voles were given the option of remaining in an empty cage or affiliating with a same-sex conspecific in a 3-h choice test.We found that early developmental mercury exposure had little if any effect on the reproductive success of breeder pairs or on affiliative behavior by either sex when subjects were tested as adults.These results suggest that,at least in the context of the behavior tested,the effects of early developmental exposure to mercury do not permanently alter adult prairie vole affiliative behavior,or do so in a way that is too subtle to be detected using the current testing paradigm.

Habitat evaluation for outbreak of Yangtze voles(Microtus fortis)and management implications

Rodent pests severely damage agricultural crops.Outbreak risk models of rodent pests often do not include sufficient information regarding geographic variation.Habitat plays an important role in rodent-pest outbreak risk,and more information about the relationship between habitat and crop protection is urgently needed.The goal of the present study was to provide an outbreak risk map for the Dongting Lake region and to understand the relationship between rodent-pest outbreak variation and habitat distribution.The main rodent pests in the Dongting Lake region are Yangtze voles(Microtus fortis).These pests cause massive damage in outbreak years,most notably in 2007.Habitat evaluation and ecological details were obtained by analyzing the correlation between habitat suitability and outbreak risk,as indicated by population density and historical events.For the source-sink population,96.18%of Yangtze vole disaster regions were covered by a 10-km buffer zone of suitable habitat in 2007.Historical outbreak frequency and peak population density were significantly correlated with the proportion of land covered by suitable habitat(r=0.68,P=0.04 and r=0.76,P=0.03,respectively).The Yangtze vole population tends to migrate approximately 10 km in outbreak years.Here,we propose a practical method for habitat evaluation that can be used to create integrated pest management plans for rodent pests when combined with basic information on the biology,ecology and behavior of the target species.

Specialist predator in a multi-species prey community:boreal voles and weasels

Dissimilar vulnerabilities of different prey types and preferences of predators are factors likely to contribute to community dynamics.This may happen via differential individual properties of prey animals(e.g.vigilance,escape)or via habitat effects making hunting by a predator easier and more rewarding in some habitats,or both.Furthermore,community dynamics may be influenced by predator mediated apparent competition,in which an increase in one prey type has negative effects on another prey type indirectly via the shared predator.We summarize the current knowledge from the field in a model predator–prey system consisting of sympatric boreal vole species and their common specialist predator and review field studies using predator manipulation and studies on the responses of individuals in the laboratory and in outdoor enclosures.The vole species studied represent different prey types that are thought to have different vulnerabilities.Our observations on the main resident specialist predator,the least weasel(Mustela nivalis nivalis L.),show that it hunts according to prey availability and suitability of the hunting habitat.Prey voles respond to the presence of the predator behaviorally in various ways to avoid predation.We conclude that even if the least weasel is a specialized predator of small rodents it acts like a generalist predator within the small rodent guild and may facilitate the coexistence of prey species via predator switching.This may lead to interspecific synchrony between prey populations,which has often been observed.We suggest that the processes determining the community impact of predator–prey interactions are driven by the behavioral arms race between the predator and the prey,together with the habitat-dependent density of prey and net gain for the predator.

Effects of maternal stress induced by predator odors during gestation on behavioral and physiological responses of offspring in Brandt’s vole(Lasiopodomys brandtii)

Maternal effects play a crucial role in regulating populations of small mammals and anti-predator behaviors of offspring.The present study investigated the effects of maternal stress induced by exposure to predator odors during gestation on the behavioral and physiological responses of offspring in Brandt’s vole(Lasiopodomys brandtii).Effects included changes in growth,behaviors,hormone levels in serum,and fosB/ΔfosB mRNA and FosB/ΔFosB protein expression in the hypothalamus.Our results showed that when pregnant voles were exposed to cat and rabbit urine odors for 18 days(1 h/day),the weight of the female offspring decreased;however,the thymus and ovary indices increased,compared with the control group.Male offspring of voles exposed to cat odor spent more time exploring and foraging,and showed a decrease in corticosterone(CORT)compared with voles in the control group.Both fosB/ΔfosB mRNA and FosB/ΔFosB protein expressions were downregulated in the hypothalamus of male offspring in the cat odor-exposed group compared to that in offspring in the control group.However,the mRNA levels of fosB/ΔfosB in the hypothalamus of female offspring did not decrease in response to cat urine odor.Our findings suggested that maternal stress induced by predator odors during gestation restrained the growth of female offspring and increased the duration of exploring and foraging behaviors of male offspring by regulating the HPA axis,which,in turn,reduced the sensitivity of offspring to unfamiliar environments and increased their probability of becoming prey.

堤岸田鼠(Bank Vole)重组朊蛋白在RT-QuIC反应上的特点及其关键位点的作用探索

堤岸田鼠对很多种属的朊病毒易感。在堤岸田鼠的朊蛋白氨基酸序列中,只有两个位点是同仓鼠和小鼠都不相同的,分别是位于C端的227位和230位。为了评估这两个位点对堤岸田鼠RT-QuIC反应的影响,我们构建了田鼠野生型（Vole-WT）,两种突变型（Vole-E227D,Vole-S230R）,以及仓鼠野生型（Ha-WT）蛋白用于RT-QuIC反应。结果显示田鼠野生型及两种突变型具有极强的RT-QuIC反应效率。即使在较弱的反应条件下,田鼠野生型及突变型也表现出比仓鼠更高的反应效率。其中Vole-E227D在三种田鼠蛋白中是效率最高的,随后为野生型和Vole-S230R。我们的结果表明堤岸田鼠朊蛋白氨基酸227位和230位不是造成堤岸田鼠RT-QuIC反应如此高的原因,但特定氨基酸位点的改变会在一定程度上影响朊蛋白的RT-QuIC反应效率。

Chevrier＇s Field Mouse(Apodemus chevrieri) and Père David＇s Vole(Eothenomys melanogaster) in China Carry Orthohepeviruses that form Two Putative Novel Genotypes Within the Species Orthohepevirus C

Hepatitis E virus(HEV)is the prototype of the family Hepeviridae and the causative agent of common acute viral hepatitis.Genetically diverse HEV-related viruses have been detected in a variety of mammals and some of them may have zoonotic potential.In this study,we tested 278 specimens collected from seven wild small mammal species in Yunnan province,China,for the presence and prevalence of orthohepevirus by broad-spectrum reverse transcription(RT)-PCR.HEV-related sequences were detected in two rodent species,including Chevrier’s field mouse(Apodemus chevrieri,family Muridae)and Père David’s vole(Eothenomys melanogaster,family Cricetidae),with the infection rates of 29.20%(59/202)and 7.27%(4/55),respectively.Further four representative full-length genomes were generated:two each from Chevrier’s field mouse(named Rd HEVAc14 and Rd HEVAc86)and Père David’s vole(Rd HEVEm40 and Rd HEVEm67).Phylogenetic analyses and pairwise distance comparisons of whole genome sequences and amino acid sequences of the gene coding regions showed that orthohepeviruses identified in Chinese Chevrier’s field mouse and Père David’s vole belonged to the species Orthohepevirus C but were highly divergent from the two assigned genotypes:HEV-C1 derived from rat and shrew,and HEV-C2 derived from ferret and possibly mink.Quantitative real-time RT-PCR demonstrated that these newly discovered orthohepeviruses had hepatic tropism.In summary,our work discovered two putative novel genotypes orthohepeviruses preliminarily named HEVC3 and HEV-C4 within the species Orthohepevirus C,which expands our understanding of orthohepevirus infection in the order Rodentia and gives new insights into the origin,evolution,and host range of orthohepevirus.