Circulation of an emerging neurotropic tick-borne phenuivirus in brown bears and wildlife in northern Japan

Circulation of an emerging neurotropic tick-borne phenuivirus in brown bears and wildlife in northern Japan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Circulation of an emerging neurotropic tick-borne phenuivirus in brown bears and wildlife in northern Japan Keita Matsuno, Anastasiia Kovba, Michito Shimozuru, Yuma Ohari, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9045918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Tick-borne phenuiviruses are a group of zoonotic pathogens causing severe, sometimes lethal, diseases in humans. While several novel phenuiviruses have been identified in ticks, especially in East Asia, their mammalian infectivity largely remains uncharacterized. Here, we report the identification and successful isolation of an emerging tick-borne phenuivirus, Toyo virus (TOYV), from Hokkaido brown bears (Ursus arctos yesoensis) during wildlife surveillance in northern Japan. TOYV was isolated from bear samples using type I and II interferon receptor-knockout (AG129) mice and Hep3B cells. Intracerebral inoculation of suckling AG129 mice induced neurological signs, and viral replication was confirmed in mouse neuroblastoma cells. Detection of TOYV RNA in brown bears and Haemaphysalis species ticks, together with seropositivity in brown bears, raccoons, and sika deer, demonstrated active tick-borne circulation of TOYV among diverse wildlife hosts. This study provides the first evidence of tick-borne phenuivirus infections in an ursid species and the neuroinvasive and neurotropic potential of TOYV. Our findings underscore the critical role of wildlife surveillance in early detection of viruses with zoonotic potential and accelerating responses to emerging viral epidemics. Biological sciences/Microbiology/Virology/Viral epidemiology Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Virology/Viral reservoirs virus brown bear phenuivirus tick-borne Japan Hokkaido wildlife Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tick-borne viruses are important pathogens of public and animal health concern, as most of them are zoonotic, meaning capable of transmitting between species [1]. Wild animals and birds can transport ticks over long distances and serve as reservoirs for tick-borne pathogens, facilitating their spread [2]. Habitat loss in wildlife populations, together with human population growth and urbanization, has led to increased contact between humans and wildlife, and consequently with wildlife-associated pathogens [1,3]. This highlights the importance of wildlife surveillance for identifying infections of emerging tick-borne pathogens that may threaten public health currently and in the near future. The family Phenuiviridae contains multiple zoonotic viruses, such as severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV), which causes an emerging disease endemic in East Asia as well as in Japan [4]. In ticks, several novel viruses belonging to the family Phenuiviridae have been detected worldwide [5,6,7,8], including Japan [9,10,11,12,13]. Some of these emerging tick-borne phenuiviruses have been shown to infect humans and animals mostly by serological assays [14,15]. However, for many other identified viruses in this family, the ability to infect mammals remains unclear. For example, Toyo virus (TOYV; genus Uukuvirus ) was first identified in Haemaphysalis formosensis ticks collected in southern Japan [13] (Fig. 1), while infection in mammals and distribution in other regions have not been reported. Because most epidemiological studies of tick-borne phenuiviruses in animals, including SFTSV, have relied on tick screening and antibody testing of animals, direct detection of viral genomes in wildlife is necessary to assess their current prevalence and host diversity. In Hokkaido, the northern island of Japan, land use changes and the expansion of agricultural lands in the past, followed by their abandonment, have created suitable habitats for some wild animal species [16]. Together with a lack of buffer zones between cities and forests, this has led to an increase in intrusions of wild animals such as feral raccoons ( Procyon lotor ), Hokkaido sika deer ( Cervus nippon yesoensis ), and Hokkaido brown bears ( Ursus arctos yesoensis ) in urban and suburban areas [17], potentially facilitating the spread of tick-borne diseases. Although rodents, birds, small mammals, and some ungulates have been relatively well studied as hosts of tick-borne pathogens in Hokkaido [18,19,20,21], data on medium- to large-sized animals remain limited. Bears, in particular, may play an important role in spreading tick-borne pathogens due to their large habitat area and long lifespans [22], but no studies have assessed the brown bear exposure to tick-borne viruses in Hokkaido. In the present study, we conducted genetic surveillance for phenuiviruses in brown bears, raccoons, and sika deer across Hokkaido, focusing on animals that intrude into human settlements. Interestingly, infections with an emerging tick-borne virus, i.e. TOYV, were identified in multiple brown bears. This represents the first report of TOYV infection in mammals, and the successful isolation of TOYV may constitute the first isolation of a tick-borne virus from ursids. We demonstrate that TOYV exhibits neuroinvasive and neurotropic potential both in vitro and in vivo . Furthermore, genetic screening of bears and ticks, together with serological surveys of wildlife, revealed endemic circulation of TOYV in Hokkaido. These findings raise concerns of TOYV zoonotic potential and its impact on animal and human health. Overall, this study highlights the importance of systematic surveillance at the wildlife–human interface, targeting diverse animal species to identify and monitor emerging tick-borne viruses. Results Identification of TOYV infection in wildlife Between 2022 and 2023, lymph node or spleen samples from raccoons and sika deer captured in Sapporo and Asahikawa, and spleen samples from brown bear captured in western (Shari town) and eastern (Rausu town) sides of the Shiretoko Peninsula (Figure 1) were tested by RT-PCR targeting multiple tick-borne viruses in the family Phenuiviridae . Nine of 149 tested bears captured in 2023 were positive, while all raccoons and sika deer were negative (Table 1). Seven sequences of the RT-PCR fragments shared 97.7% nucleotide similarity with the TOYV RNA-dependent RNA polymerase (RdRp) gene, and two partial sequences with a high number of ambiguities showed 88.0% and 89.2% similarity (Supplementary Table 4). TOYV isolation and characterization The amounts of TOYV RNA in PCR-positive bear tissues were quantified. Most bears had detectable viral RNA in the liver and spleen, and a few in plasma and lung samples (Figure 2A). For isolation of TOYV, bear samples with high virus load were administered intraperitoneally to adult AG129 mice. While no weight loss or clinical signs were observed, viral RNA was detected in tissues at 21 dpi, with the highest quantities in spleen and brain (Figure 2B). A brain homogenate sample with the highest viral load from the mouse (infected with samples of bear ID: 23B51) was then inoculated into mammalian cell lines for the in vitro isolation. No CPE or continuous viral growth could be observed in Vero E6, BHK21, and Huh-7 cells (data not shown). Productive viral growth resulting in cell death was detected only in Hep3B cells (Figure 2C), and viral antigen expression was confirmed by IFA using immunized mouse serum as primary and anti-mouse IgG as secondary antibodies (Figure 2D). The TOYV isolate (named 23B51b) replicated efficiently in Hep3B (Figure 2E). Electron microscopy of the supernatant of infected Hep3B cells revealed spherical particles of approximately 90 nm, matching the morphology of viruses in the genus Uukuvirus (Figure 2F). Neuroinvasive potential and neurotropism of TOYV To confirm the replication of TOYV in the brain, nine newborn AG129 mice were intracerebrally inoculated with the brain sample used for virus isolation (Figure 3A). Neurological signs, including ataxia, delayed righting reflex, and impaired movements, developed between 11 and 13 dpi and were accompanied by a marked increase in viral RNA levels in brain tissue (Figure 3B). The neurotropism of the TOYV isolate in neural tissue was further assessed using the mouse neuroblastoma cell line Neuro-2a (Figure 3A). Viral RNA levels increased over time without showing CPE (Figure 3C), with virus titer reaching 2x10 4 TCID 50 /ml at 8 dpi. Viral antigen was also detected in infected cells by IFA (Figure 3D). The neuroinvasive potential of TOYV was then assessed by intraperitoneal inoculation of the TOYV isolate 23B51b into adult AG129 and ICR mice (Figure 3A). Although all inoculated AG129 and ICR mice showed detectable viral loads in their spleens, virus in the brain was detected in only one AG129 mouse (Figure 3E). TOYV screening in ticks To determine a potential vector species of ticks for TOYV, ticks collected on the Shiretoko Peninsula in 2024 and 2025 were pooled by species and developmental stage into a total of 102 pools and screened for TOYV using RT-qPCR, with all samples testing negative (Supplementary Table 5). A retrospective analysis of archived tick metagenomes was performed, detecting TOYV genomes in a pool of Haemaphysalis megaspinosa nymphs collected in April 2015 and in a pool of Haemaphysalis flava females collected in October 2018 from central Hokkaido (previously reported in GenBank as LC604128). TOYV genome sequencing and phylogenetic analysis Coding sequences of the RdRp, the glycoprotein precursor (Gn and Gc), and nucleocapsid (N) and nonstructural (NSs) proteins encoded by the L, M, and S segment RNAs, respectively, were obtained from tissue samples of six of the RT-PCR–positive brown bears by using primers designed for the TOYV reference. Complete sequences of coding regions of TOYV in the archived H. flava pool (18THF) were also obtained by metagenome sequencing. Phylogenetic analyses showed that all available TOYV sequences across three genomic segments were closely related and clustered into a highly supported clade with the corresponding TOYV sequences available in GenBank, which were obtained from H. formosensis ticks collected in Ehime Prefecture, Japan (Figure 4A-D). The TOYV cluster was included in the Kaisodi group, which is consistent with previous reports. Additional analysis within the TOYV sequences group, including a partial sequence obtained by a metagenome sequencing of a H. megaspinosa tick pool (15THM), showed that TOYVs from bears 23B53 and 23B73 clustered together with 15THM, while sequences from bears 23B51, 23B55, 23B65, and 23B72 formed a separate cluster (Figure 4E-F). TOYV screening in wildlife in Hokkaido First, to examine the infections of TOYV among bears, bear tissues collected in 2024 were subjected to RT-PCR, and those collected in 2025 to RT-qPCR for genetic screening. None of the 19 bears in 2024 tested positive, while six of 60 animals were positive in 2025 (Supplementary Table 6). To further assess TOYV exposure in brown bears and other wildlife species in Hokkaido, serum samples from 208 brown bears, 161 sika deer, 210 raccoons, and 187 rodents were subjected to serological screening using ELISA, with positive results confirmed by IFA (Table 2). Among brown bears, seven out of 77 (9.1%) in Shari and two out of 50 (4%) in Rausu were seropositive in 2023. In total, ten out of 64 in 2025 tested positive, while 17 captured in 2024 were negative. All ELISA-positive bears in 2023 and 2025, as well as all PCR-positive bears in 2025, were captured in autumn, 22 of 28 (78.6%) being females (Table 3) among 126 females out of 208 captured bears (60.6%). Notably, a few cubs that were born in 2025 were found infected (Table 3). A seasonal difference in sampling was observed, with a larger number of animals tested in autumn (Figure 5A). Among sika deer, none tested positive in Shiretoko National Park or northern Hokkaido (Asahikawa), whereas a few captured in central Hokkaido (Sapporo) were seropositive (Table 2). However, sample sizes varied by region and season (Figure 5B). While none of the raccoons captured in Asahikawa were positive, seroprevalence in Sapporo consistently exceeded 25% between 2022 and 2024, with positive animals detected from spring through autumn (Table 2; Figure 5C). All tested rodents captured in central Hokkaido were seronegative (Table 2). Discussion In this study, we performed genetic screening of tick-borne phenuiviruses in wildlife in Hokkaido, Japan, and identified brown bear infections with TOYV, a phenuivirus previously reported in ticks in Japan [13]. TOYV was successfully isolated, likely representing the first recovery of a tick-borne virus from any ursid species in the world, as studies of viral infections in ursids remain limited [23,24]. Moreover, our findings imply the neuroinvasive and neurotropic potential of TOYV actively circulating within Japan, which may pose risks to impact animal and human health. We successfully isolated TOYV from the brain tissue of mice inoculated with bear samples intraperitoneally, suggesting that the virus has neuroinvasive potential. This was further supported by the observations in vitro and in vivo , particularly with neurological signs and active viral replication in the brain tissue of infected newborn mice and the detection of viral RNA in the brains of adult AG129 mice following intraperitoneal infection with the virus isolate. Since virus detection in the mouse brain was observed only in interferon-receptor knockout AG129, but not in inbred wild-type ICR mice, the host innate immune response plays an important role in protecting the central nervous system from TOYV infection, as reported for other neurotropic viruses [25,26]. This study may represent the first report of neuroinvasive potential among uukuviruses, including Kaisodi group phenuiviruses, to which TOYV belongs, which were shown to infect mammals and birds [27-29]. Neurological diseases, as well as virus detection in cerebrospinal fluid, were observed in patients infected with human pathogenic tick-borne phenuiviruses, such as SFTSV and Lone Star virus [30,31], raising the possibility that neurotropism may extend to multiple members of the Phenuiviridae family and underscoring the need for further investigation of TOYV neurovirulence. Our wildlife surveillance revealed infections of TOYV in multiple species of mammals as well as ticks in Hokkaido, distant from the place where TOYV was initially identified in ticks [13]. Hokkaido is located on the East Asian–Australasian Flyway, and the Shiretoko Peninsula serves as a stopover for multiple migratory bird species that can introduce ticks from other regions of Japan or overseas [18,32]. Thus, TOYV may have been introduced to Hokkaido or vice versa via migratory birds. TOYV derived from brown bears and ticks phylogenetically showed no clear geographic clustering by sampling location, suggesting viral circulation within Hokkaido and also Japan, likely facilitated by long-distance transportation of ticks by birds. TOYV-infected brown bears were predominantly female and captured on the western side of the Shiretoko Peninsula (Shari) during autumn. Since female bears’ typical home ranges are less than 30 km², much smaller than those of male bears [33], TOYV infections likely occurred within limited areas of the Shiretoko Peninsula. The geographical distribution of TOYV in bears may therefore be influenced by the locations of migratory birds' stopover sites, as well as by differences in tick species composition between the two coasts of the peninsula, which are separated by a mountain range that shapes the regional climate [34]. TOYV was initially detected in H. formosensis in southern Japan [13] and retrospectively detected in H. megaspinosa ticks and H. flava ticks collected in the central Hokkaido long before the detection of infection in brown bears in Shiretoko. Viruses in the Kaisodi group are known to be transmitted by Haemaphysalis ticks [29], and our findings strongly suggest the involvement of Haemaphysalis ticks in TOYV circulation. We also revealed that the seroprevalence of TOYV in raccoons and sika deer was different among regions in Hokkaido, and similar to the observation of the TOYV-positive bears, the infected animals were predominantly found in autumn. These regional and seasonal occurrences of TOYV infections may be determined by the abundance and seasonality of Haemaphysalis ticks, as well as regional and seasonal differences in the host preference of ticks [35,36]. Further understanding of the vector and host competencies of each tick and animal species for TOYV will help to reveal the mechanism behind the emergence of tick-borne phenuiviruses. In summary, we report the infections of the emerging phenuivirus, potentially pathogenic to animals and humans, in wildlife in Hokkaido, Japan, and demonstrate the TOYV active circulation in the region. This underscores the need for further investigation of TOYV pathogenicity and continued monitoring to assess its spread in animals and potentially humans. Due to the limited availability of wildlife samples, we could not examine TOYV infections in the brains of wildlife in the present study, and thus, the impact of TOYV spread on wildlife and public health should be carefully investigated. This study emphasizes that wild animals are important targets for monitoring the emergence of novel tick-borne viral infections and supports the integration of systematic wildlife pathogen screening into national health programs. Materials and Methods Sample collection from wildlife Samples of sika deer and raccoons from central (Sapporo) and northern (Asahikawa) Hokkaido, as well as serum samples of live-captured Hokkaido sika deer within Shiretoko National Park, were collected as part of the previous study [37]. Brown bears outside the Shiretoko National Park are culled annually for management purposes and used for sampling on both the eastern and western coasts, which are separated by a mountain range (Figure 1). Spleen, liver, lung, and blood samples were collected from the brown bears between March 2023 and October 2025. Samples were stored at −20°C prior to delivery to Hokkaido University for further processing. The brown bear age was estimated based on body size, observation of canine tooth development, and the presence of the mother at the time of capture, and is reported as <1 year for dependent cubs assumed to have been born in the year of capture [38]. Rodent trapping was performed in Central Hokkaido (Figure 1) during May 2025 to June 2025 using Sherman traps, and captured rodents were euthanized under isoflurane anesthesia, followed by cervical dislocation. Blood was collected directly from the heart and centrifuged to separate serum, followed by storage at –20°C until use. RNA extraction Total RNA of animal spleen and lymph node tissue samples was extracted using NucleoSpin RNA Kit (TAKARA Bio Inc.). Briefly, tissues initially stored at −20 °C were cut into smaller pieces and re-frozen at −80 °C prior to RNA extraction. For extraction, lysis buffer supplemented with carrier RNA was added directly to the frozen tissue, and samples were homogenized at 3,000 rpm for 1 min at 4°C. The lysates were then filtered through a NucleoSpin Filter, and RNA extraction was performed according to the manufacturer’s instructions. The RNA was eluted in 50-60 µL of RNase-free water and stored at –80°C. RT-PCR and Sanger sequencing One-step RT-PCR was performed with primers targeting the conserved region of the genome among multiple viruses in Phenuiviridae family, as described previously [12]. Amplified partial genomes were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions in the 3500xL Genetic Analyzer (Applied Biosystems). Obtained sequences were compared to the sequences in the NCBI database. RT-qPCR Primers and a tag-labeled probe targeting TOYV S segment were designed using the IDT PrimerQuest Tool (Integrated DNA Technologies) (Supplementary Table 1). The assay was performed with One Step PrimeScript III RT-qPCR Mix (TAKARA Bio Inc.). A standard curve was generated using 10-fold serial dilutions of DNA amplified with the same primers, with a detection limit of 40 copies/well. The thermal profile included 25°C for 10 min, 52°C for 2 min, followed by 45 cycles of 95°C for 10 sec and 60°C for 30 sec, and was performed on a qTOWER3 Real-time PCR Thermal Cycler (Analytik Jena AG). All the samples were measured in duplicate, and samples with the cycle threshold (Ct) less than 42 in at least one replicate were considered positive. Mouse infection experiments for in vivo v irus isolation and characterization Type I and II interferon receptor-knockout (AG129) mice were in-house bred (obtained from Marshall BioResources), and ICR inbred mice were obtained from Japan SLC. Experimental design for animal experimentation is indicated in Figure 3A. For virus isolation using mice, samples (i.e., plasma and/or tissue homogenates) from RT-PCR-positive brown bears were inoculated intraperitoneally into adult AG129 mice. Animals were monitored daily, and internal organs were collected for RNA quantification with RT-qPCR. The brain sample from an AG129 mouse with the highest viral load was used for further passaging and for intracerebral inoculation into a litter of 2-day-old (d.o.) AG129 pups. Three asymptomatic pups inoculated with the adult AG129 brain sample were euthanized at 7 dpi, and one at 21 dpi. Five pups that developed neurological signs were euthanized at 11–13 dpi. The brain tissues of pups were collected for viral RNA quantification. For examining the pathogenesis of TOYV infections, especially neurological signs, four adult ICR and three adult AG129 were intraperitoneally inoculated with 3,000 TCID 50 of TOYV isolate. Animals were monitored daily and euthanized at 20 dpi. Brain and spleen tissues were collected for viral RNA quantification. Cell culture Vero E6 (African green monkey kidney), BHK21 (Syrian hamster kidney), Huh-7 (human hepatoma), and Hep3B (human liver carcinoma) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Nacalai Tesque Inc.) supplemented with 10% Fetal Bovine Serum (FBS) (ICN Biomedicals Inc.), 1% Penicillin–Streptomycin (Thermo Fisher Scientific), and 1% L-glutamine (FUJIFILM Wako Chemicals). Neuro-2a (mouse neuroblastoma) cells were cultured in Eagle's Minimum Essential Medium (EMEM) (FUJIFILM Wako Chemicals) supplemented with 1% MEM Non-Essential Amino Acids Solution (NEAA) (Thermo Fisher Scientific), 10% FBS, and 1% Penicillin–Streptomycin. Following infection, all cells were maintained in medium containing 2% FBS. Virus isolation in vitro AG129 mouse brain tissue was homogenized in DMEM supplemented with 10% FBS and centrifuged. The supernatant was inoculated onto VeroE6, BHK21, Huh-7, and Hep3B cell monolayers in T25 culture flasks. After 1 h of incubation at 37 °C, cells were washed with PBS and overlaid with DMEM containing 2% FBS. Maximum three passages (4–7 days each) were performed. Cells were monitored for cytopathic effects (CPE), and viral RNA in the supernatant was quantified by TOYV-specific RT-qPCR. Infection was confirmed by immunofluorescence assay using mouse convalescent serum as the primary antibody. Transmission electron microscopy (TEM) Supernatant from Hep3B cells infected with TOYV was centrifuged at 3,500 rpm for 10 min at 4°C. The virus was then pelleted down through a 20% sucrose cushion by ultracentrifugation at 28,000 rpm for 2 h at 4°C using an SW32Ti rotor in an Optima L-90K centrifuge (Beckman Coulter). The resulting pellet was resuspended in 100 µL of PBS and stored overnight at 4°C. Concentrated virions were placed onto carbon-coated, ion-sputtered grids (Nisshin EM) for 1 min and negatively stained with 2% phosphotungstic acid (PTA) (pH 7.0) for 15 s. Transmission electron microscopy was performed using a Hitachi H-7650 instrument (Hitachi High-Tech). Enzyme-linked immunosorbent assay (ELISA) Hep3B cells infected with TOYV were incubated with lysis buffer at 4°C for 30 min (ATTO co., ltd). The samples were centrifuged at 15,000 rpm for 15 min at 4°C, supernatants collected and stored at –80°C till use. The lysate of cells infected with TOYV was used as ELISA antigen, with uninfected cell lysate as a negative control antigen. Lysates were diluted in PBS, coated onto plates, and incubated overnight at 4°C. After a single tris-buffered saline with Tween 20 (TBS-T) wash, 50 µL of plasma or serum (1:400 diluted in Blocking One (Nacalai Tesque Inc.)) was added to each well and incubated for 1 h at room temperature. Following three washes, HRP-conjugated Protein A/G (32490, Thermo Fisher Scientific) (for sika deer, raccoon, and brown bear samples) or HRP-conjugated anti-mouse antibodies (ab6789, Abcam) (for rodent samples) were applied, followed by incubation at room temperature for 1 h. Plates were washed again, developed with 100 µL 3,3',5,5'-tetramethylbenzidine (TMB) substrate for 15-20 min at room temperature, and the reaction was stopped with 100 µL of 1M HCl. Optical density (OD) was measured at 450 nm in a plate reader (Multiskan FC). OD difference (∆OD) between the antigen-coated well and the negative control cell lysate-coated well was calculated, and the cutoff value was measured according to the following formula: Negative samples were defined as brown bear sera samples collected in 2023 that tested negative by immunofluorescent assay (IFA, see below) at a 1:50 dilution. Based on this calculation, the cutoff value was set at 0.3 (all ∆OD are provided in Supplementary Figure 1). Notably, for raccoon sera, several samples with ∆OD between 0.3 and 0.5 were found to be IFA-negative. Therefore, to maximize diagnostic specificity and reduce the risk of false-positives, the cutoff for raccoons was retrospectively increased to 0.5 (a breakdown of ∆OD for raccoons based on IFA results is provided in Supplementary Figure 2). Sera samples tested as positive in ELISA were examined by IFA as described below. IFA 96-well flat-bottom plates were coated with e-poly-L-lysine (1:10 in PBS), incubated at 37°C for 30 min. Hep3B cells were then seeded into the plates, grown to monolayers, and infected with 10 TCID 50 (50% tissue culture infectious dose) of TOYV isolate. After 4 days of culture, cells were fixed with 4% paraformaldehyde (PFA) (FUJIFILM Wako Chemicals) for 1 h at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 (SIGMA-ALDRICH) for 15 min, then washed again. Animal sera or plasma samples were heat-inactivated at 56°C for 30 min and diluted 1:50 in culture medium. 50 µL of diluted sample was added per well and incubated for 1 h at room temperature. After three PBS washes, cells were incubated with FITC-conjugated Protein A/G (BioVision), then nuclei were counterstained with DAPI (FUJIFILM Wako Chemicals) and images taken with a fluorescence microscope (ZEISS). For mouse sera, Alexa Fluor 488–labeled anti-mouse IgG (A32723 or A-11029, Invitrogen, Thermo Fisher Scientific), and for raccoons, with high background staining, anti-raccoon HRP-conjugated IgG (A140-123P, Bethyl Laboratories) were used as the secondary antibodies. TOYV survey in ticks In 2024 and 2025, ticks were collected on the Shiretoko Peninsula using flannel flags during spring-early summer (April, May, and June) and autumn (September and October). Specimens were identified morphologically [39], and total DNA and RNA were extracted from individual ticks or tick pools (up to 10 individuals of the same sex and developmental stage) using the BlackPrep Tick DNA/RNA kit (Analytik Jena) following the manufacturer’s protocol. Tick RNA was screened for TOYV using RT-qPCR as described above. In addition, a retrospective analysis of TOYV was performed using a BLASTn search of assembled sequences from in-house metagenomic RNA-seq data derived from ticks collected in Hokkaido [40], as well as a BLASTn search of the GenBank database. Additional sequencing and viral genome assembly were performed as described below. TOYV genome sequencing and phylogenetic analysis For determining the full-genome sequences of TOYVs from brown bears, the RT-PCR positive bear spleen and liver RNAs were used for cDNA synthesis with the PrimeScript II 1 st Strand cDNA Synthesis Kit (TAKARA Bio Inc) following the manufacturer’s instructions. The cDNA was used as a template for PCR amplification of TOYV genome fragments with primers designed based on TOYV reference sequences in GenBank (Accession numbers: LC618931, LC618932, LC618933). Amplified DNA was sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit with additional customized primers. To assemble TOYV genomes from two TOYV-positive tick pools determined during the retrospective virus genome screening, paired-end Illumina NextSeq reads were quality-checked and trimmed to remove adapter sequences and low-quality bases. Filtered reads were then mapped to a reference TOYV genome retrieved from GenBank using standard alignment tools in Geneious Prime software version 2025.2.2 (https://www.geneious.com), and consensus sequences were generated from the mapped reads. All TOYV sequences obtained in this study are deposited in the GenBank database (their accession numbers are listed in the Supplementary Table 2). The obtained sequences were aligned with the corresponding TOYV genome and representative members of the family Phenuiviridae (accession numbers provided in Supplementary Table 3). Multiple sequence alignment of the nucleotide sequence for four protein coding genes (RdRp, G, N and NSs) was performed using the Translation Align function in the Geneious Prime software. Alignment curation was performed manually by removing gap-containing regions while preserving the codon structure of the coding sequences. Maximum-likelihood phylogenetic trees for four genes were constructed with IQ-TREE 2 [41] with nucleotide substitution models selected by ModelFinder [42] under the -m MFP option based on partitioned codon positions. Node support was assessed using 10,000 replicates of the Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) [43] and ultrafast bootstrap approximation (UFboot) [44]. Declarations Ethical statement All animal experiments were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The study was approved by the Animal Care and Use Committee of Hokkaido University under permits No. 22-0016, No. 18-0001, No. 18-0083, No. 23-0014, No. 23-0178, and No. 25-0119. Animal infection experiments were carried out in the biosafety level 2 facility at the Hokkaido University International Institute for Zoonosis Control. Permission for rodent capture for academic purposes was obtained from the Hokkaido Government. Acknowledgements We appreciate the Shiretoko Nature Foundation staff and members of the Wildlife Biology and Medicine Laboratory at Hokkaido University for their assistance in the sample collection. We appreciate the members of the Division of Risk Analysis and Management at the International Institute for Zoonosis Control, especially Asako Aoshima/Shigeno, for the technical assistance. We also appreciate Soyo Ohsako, Yuki Shirota, Kenya Suzuki, Yuta Tsukahara, and Natsuki Miyazaki from the Faculty of Veterinary Medicine at Hokkaido University for their contribution to tick collection. Author Contributions A.K. – study design and implementation, sample acquisition, data analysis, funding acquisition, and manuscript preparation. M.S. – management, funding acquisition, and sample acquisition. Y.O. – data analysis and visualization. M.I. – sample and funding acquisition. J.L. and Y.M. – study implementation. Kh.S., T.K., M.Y., Ko.S., and L.U. – sample acquisition. Y.O., M.S., N.K., E.F., R.N., M.L.K., and Y.T. – sample acquisition and expertise sharing. T.T. – study design and supervision, funding acquisition. K.M. – study design and implementation, expertise sharing, and funding acquisition. All authors reviewed and approved the final manuscript. Competing interest statement The authors declare no competing interests. References Jones, K. E. et al. Global trends in emerging infectious diseases. Nature 451 , 990–993 (2008). Baneth, G. Tick-borne infections of animals and humans: a common ground. Int. J. Parasitol. 44 , 591–596 (2014). Hassell, J. M. et al. Urbanization and disease emergence: dynamics at the wildlife-livestock-human interface. Trends Ecol. Evol. 32 , 55–67 (2017). Silvas, J. A. & Aguilar, P. V. The emergence of severe fever with thrombocytopenia syndrome virus. Am. J. Trop. Med. Hyg. 97 , 992–996 (2017). Li, C. X. et al. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. eLife 4 , e05378 (2015). Brinkmann, A. et al. A metagenomic survey identifies Tamdy orthonairovirus as well as divergent phlebo-, rhabdo-, chu- and flavi-like viruses in Anatolia, Turkey. Ticks Tick Borne Dis. 9 , 1173–1183 (2018). Koka, H. et al. Detection and prevalence of a novel Bandavirus related to Guertu virus in Amblyomma gemma ticks and human populations in Isiolo County, Kenya. PLoS One 19 , e0310862 (2024). Shen, S. et al. A novel tick-borne phlebovirus, closely related to severe fever with thrombocytopenia syndrome virus and Heartland virus, is a potential pathogen. Emerg. Microbes Infect. 7 , 95 (2018). Ejiri, H. et al. Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan. Virus Res. 244 , 252–261 (2018). Matsuno, K. et al. The unique phylogenetic position of a novel tick-borne phlebovirus ensures an ixodid origin of the genus Phlebovirus . mSphere 3 , e00239-18 (2018). Mekata, H., Kobayashi, I. & Okabayashi, T. Detection and phylogenetic analysis of Dabieshan tick virus and Okutama tick virus in ticks collected from Cape Toi, Japan. Ticks Tick Borne Dis. 14, 102237 (2023). Matsuno, K. et al. Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus. J. Virol. 89, 594–604 (2015). Kobayashi, D. et al. Toyo virus, a novel member of the Kaisodi group in the genus Uukuvirus (family Phenuiviridae ) found in Haemaphysalis formosensis ticks in Japan. Arch. Virol. 166, 2751–2762 (2021). Tran, N. T. B. et al. Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan. J. Vet. Med. Sci. 84, 82–89 (2022). Torii, S. et al. Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles. Ticks Tick Borne Dis. 10, 328–335 (2019). Ohashi, H. et al. Land abandonment and changes in snow cover period accelerate range expansions of sika deer. Ecol. Evol. 6, 7763–7775 (2016). Hokkaido Government Environmental Bureau. https://www.pref.hokkaido.lg.jp/ks/skn/ (accessed 3 December 2025). Nishino, A. et al. Transboundary movement of Yezo virus via ticks on migratory birds, Japan, 2020–2021. Emerg. Infect. Dis. 30, 2674–2678 (2024). Jamsransuren, D. et al. Epidemiological survey of tick-borne encephalitis virus infection in wild animals on Hokkaido and Honshu Islands, Japan. Jpn. J. Vet. Res. 67, 163–172 (2019). Sashika, M. et al. Molecular survey of rickettsial agents in feral raccoons ( Procyon lotor ) in Hokkaido, Japan. Jpn. J. Infect. Dis. 63, 353–354 (2010). Kodama, F. et al. A novel nairovirus associated with acute febrile illness in Hokkaido, Japan. Nat. Commun. 12, 5539 (2021). Tiffin, H. S., Skvarla, M. J. & Machtinger, E. T. Tick abundance and life-stage segregation on the American black bear ( Ursus americanus ). Int. J. Parasitol. Parasites Wildl. 16, 208–216 (2021). Shi, Y. et al. Extensive cross-species transmission of pathogens and antibiotic resistance genes in mammals neglected by public health surveillance. Cell 188, 6591–6605.e14 (2025). Alex, C. E. et al. Viruses in unexplained encephalitis cases in American black bears ( Ursus americanus ). PLoS One 15, e0244056 (2020). Milora, K. A. & Rall, G. F. Interferon control of neurotropic viral infections. Trends Immunol. 40, 842–856 (2019). Chotiwan, N. et al. Type I interferon shapes brain distribution and tropism of tick-borne flavivirus. Nat. Commun. 14, 2007 (2023). Palacios, G. et al. Characterization of the Uukuniemi virus group ( Phlebovirus: Bunyaviridae ): evidence for seven distinct species. J. Virol. 87, 3187–3195 (2013). Hoff, G. L. et al. Isolations of Silverwater virus from naturally infected snowshoe hares and Haemaphysalis ticks from Alberta and Wisconsin. Am. J. Trop. Med. Hyg. 20, 320–325 (1971). International Committee on Taxonomy of Viruses. https://ictv.global/report/chapter/phenuiviridae/phenuiviridae/uukuvirus (accessed 28 January 2026). Shan, D. et al. Severe fever with thrombocytopenia syndrome with central nervous system symptom onset: a case report and literature review. BMC neurology 24, 158 (2024). Chiu, CY. et al. Two Human Cases of Fatal Meningoencephalitis Associated with Potosi and Lone Star Virus Infections, United States, 2020-2023. Emerging infectious diseases 31, 215-221 (2025). Nakao, R. et al. Amblyomma testudinarium infestation on a brown bear ( Ursus arctos yesoensis ) captured in Hokkaido, a northern island of Japan. Parasitol. Int. 80, 102209 (2021). Shimozuru, M. et al. Estimation of breeding population size using DNA-based pedigree reconstruction in brown bears. Ecol. Evol. 12, e9246 (2022). Shiretoko Nature Foundation. https://www.shiretoko.or.jp/report/2023/09/7579.html (accessed 3 December 2025). Shimizu, K. et al. Seasonal infestation patterns of ticks on Hokkaido sika deer ( Cervus nippon yesoensis ). Parasitology 151, 1317–1325 (2024). Ito, M. et al. Environmental and host factors underlying tick infestation in invasive raccoons ( Procyon lotor ) in Hokkaido, Japan. Ticks Tick Borne Dis. 15, 102389 (2024). Kovba, A. et al. No evidence of SARS-CoV-2 infection in urban wildlife of Hokkaido, Japan. Transbound. Emerg. Dis. 2024, 1204825 (2024). Shimozuru, M. et al. Reproductive parameters and cub survival of brown bears in the Rusha area of the Shiretoko Peninsula, Hokkaido, Japan. PLoS One 12, e0176251 (2017). Yamaguti, N. et al. Ticks of Japan, Korea, and the Ryukyu Islands. Brigham Young Univ. Sci. Bull. Biol. Ser. 15, 1 (1971). Torii, S. et al. Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles. Ticks Tick Borne Dis . 10, 328-335 (2019). Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 2461 (2020). Kalyaanamoorthy, S. et al. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017). Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010). Hoang, D. T. et al. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018). Tables Table 1. Phenuivirus RT-PCR screening in Hokkaido wildlife. Animal Year Tissue RT-PCR (positive/tested) Raccoon 2022 Lymph node 0/94 2023 Spleen 0/100 Sika deer 2022 Lymph node 0/7 2023 Spleen 0/28 Brown bear 2023 Spleen 9/149 Table 2. Antibody screening for TOYV in wild animals inhabiting Hokkaido, Japan (2022-2025). Order Species (ENG) Species (Latin) Location Year Tested Seroprevalence Subtotal IFA+/tested (%) Total IFA+/tested (%) ELISA + IFA + Antibody prevalence % by IFA Carnivora Hokkaido brown bear Ursus arctos lasiotus Shari 2023 77 9 7 9.1 13*/131 (9.9) 19/208 (9.1) 2024 14 0 0 0 2025 40 8 6 15 Rausu 2023 50 2 2 4 6**/77 (7.8) 2024 3 0 0 0 2025 24 4 4 16.7 Raccoon Procyon lotor Sapporo 2022 55 14 14 25.5 58/179 (32.4) 58/210 (27.6) 2023 73 32 29 39.7 2024 51 16 15 29.4 Asahikawa 2022 24 0 0 0 0/31 (0.0) 2023 7 0 0 0 Artiodactyla Hokkaido sika deer Cervus nippon yesoensis Sapporo 2022 5 0 0 0 2/44 (4.6) 2/161 (1.2) 2023 19 3 1 5.3 2024 10 6 0 0 2025 10 2 1 10 Asahikawa 2022 2 0 0 0 0/82 (0.0) 2023 45 0 0 0 2024 35 0 0 0 Shiretoko National Park 2023 35 0 0 0 0/35 (0.0) Rodentia Large Japanese field mouse Apodemus speciosus Central Hokkaido 2025 60 0 0 0 0/60 (0.0) 0/187 (0.0) Japanese red-backed vole Craseomys rufocanus bedfordiae Central Hokkaido 2025 52 0 0 0 0/52 (0.0) Small Japanese field mouse Apodemus argenteus Central Hokkaido 2025 69 0 0 0 0/69 (0.0) Japanese Northern red-backed Vole Clethrionomys rutilus mikado Central Hokkaido 2025 6 0 0 0 0/6 (0.0) *Including 5 RT-PCR-positive bears. **Including 1 RT-PCR-positive bear. Table 3 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files 20260306SupplementaryFiles.pdf Supplementary Files Table3.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9045918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":607578910,"identity":"0ebf7bd7-9a10-4c4f-bd83-41e5c51e2ed7","order_by":0,"name":"Keita Matsuno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYNCCAzYgkg3IYGBsYGaAcnACkIoDaaRrOYykhZCTdNvPH/7Mc+a8vHz7AbYHH84clu1vZ2D88IOBLw+XFrMzyWzSPDduGzb2JLAbzrhx2HjGYQZmyR4GtmKcWg4kszHzfLidwCzBANT74XBiA9CR0kBXJuJyodn5x8yfeT6cS2CDaZkPtOU3Xi03khmADjuQwAPWcuNw4obDQAZ+LY/NJOecSTacwZPYJjnjTLrxxsOMbZY9Bnj8cj7x8Yc3x+yAIXb4mMSHY9ay884fPnzjR8UxnCGGBOAxAmIYHEsgQgsqqCFdyygYBaNgFAxXAACQhli5NOK63QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4205-6526","institution":"Hokkaido University, International Institute for Zoonosis Control","correspondingAuthor":true,"prefix":"","firstName":"Keita","middleName":"","lastName":"Matsuno","suffix":""},{"id":607578911,"identity":"0a6112ab-290b-4f1e-b799-fc445bdd162f","order_by":1,"name":"Anastasiia Kovba","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Anastasiia","middleName":"","lastName":"Kovba","suffix":""},{"id":607578912,"identity":"b5954320-c8fb-44ec-9c88-10a8d9a4e29d","order_by":2,"name":"Michito Shimozuru","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Michito","middleName":"","lastName":"Shimozuru","suffix":""},{"id":607578913,"identity":"b190f652-27bf-474f-85c3-d1b32133f38e","order_by":3,"name":"Yuma Ohari","email":"","orcid":"https://orcid.org/0000-0003-3008-3102","institution":"Hokkiado University","correspondingAuthor":false,"prefix":"","firstName":"Yuma","middleName":"","lastName":"Ohari","suffix":""},{"id":607578914,"identity":"a92f6ce3-8688-4ea0-b857-bba42a30d1aa","order_by":4,"name":"Mebuki Ito","email":"","orcid":"","institution":"Hokkaido University, International Institute for Zoonosis Control","correspondingAuthor":false,"prefix":"","firstName":"Mebuki","middleName":"","lastName":"Ito","suffix":""},{"id":607578915,"identity":"b864652a-1a10-42e9-8ae1-70f9a24ece90","order_by":5,"name":"Jingshu Li","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Jingshu","middleName":"","lastName":"Li","suffix":""},{"id":607578916,"identity":"2a0b66c1-e9ae-4827-8fef-3ec1d362084b","order_by":6,"name":"Yume Mimura","email":"","orcid":"","institution":"Hokkaido University, International Institute for Zoonosis Control","correspondingAuthor":false,"prefix":"","firstName":"Yume","middleName":"","lastName":"Mimura","suffix":""},{"id":607578917,"identity":"dfa1831f-4035-41d7-95ad-54fe07918110","order_by":7,"name":"Kohei Shinjo","email":"","orcid":"","institution":"Shiretoko Nature Foundation","correspondingAuthor":false,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Shinjo","suffix":""},{"id":607578918,"identity":"d0c793f6-f400-4e5a-a272-99a27feda899","order_by":8,"name":"Teruhiro Kanagawa","email":"","orcid":"","institution":"Shiretoko Nature Foundation","correspondingAuthor":false,"prefix":"","firstName":"Teruhiro","middleName":"","lastName":"Kanagawa","suffix":""},{"id":607578919,"identity":"da3e0a53-2f7f-46dd-a3ff-705eea338864","order_by":9,"name":"Masami Yamanaka","email":"","orcid":"","institution":"Shiretoko Nature Foundation","correspondingAuthor":false,"prefix":"","firstName":"Masami","middleName":"","lastName":"Yamanaka","suffix":""},{"id":607578920,"identity":"44d54783-e6de-406c-a242-7a246bc942ad","order_by":10,"name":"Kotaro Shimizu","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Kotaro","middleName":"","lastName":"Shimizu","suffix":""},{"id":607578921,"identity":"ec2e4e53-2c85-4a6e-a174-1cb96e9f9241","order_by":11,"name":"Leo Uchida","email":"","orcid":"","institution":"Rakuno Gakuen University","correspondingAuthor":false,"prefix":"","firstName":"Leo","middleName":"","lastName":"Uchida","suffix":""},{"id":607578922,"identity":"1afe1ae9-7966-4e9e-902e-a8c41b18c94d","order_by":12,"name":"Yasuko Orba","email":"","orcid":"https://orcid.org/0000-0001-9910-3912","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Yasuko","middleName":"","lastName":"Orba","suffix":""},{"id":607578923,"identity":"1162bfd5-1d70-4855-845e-85f6f8a2a3dd","order_by":13,"name":"Michihito Sasaki","email":"","orcid":"https://orcid.org/0000-0003-1607-2175","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Michihito","middleName":"","lastName":"Sasaki","suffix":""},{"id":607578924,"identity":"dbb4932d-9364-4007-946b-cbdcab5b50e5","order_by":14,"name":"Nijiho Kawaguchi","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Nijiho","middleName":"","lastName":"Kawaguchi","suffix":""},{"id":607578925,"identity":"06a126cb-1234-4dce-b591-6bc2092d7050","order_by":15,"name":"Eri Fujii","email":"","orcid":"","institution":"Hokkaido University, International Institute for Zoonosis Control","correspondingAuthor":false,"prefix":"","firstName":"Eri","middleName":"","lastName":"Fujii","suffix":""},{"id":607578926,"identity":"f85c300c-5e9d-494d-9403-f79121029276","order_by":16,"name":"Ryo Nakao","email":"","orcid":"https://orcid.org/0000-0002-3105-7603","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Nakao","suffix":""},{"id":607578927,"identity":"8f779d50-7371-4fc1-a127-55006d4e8bc6","order_by":17,"name":"Mackenzie Kwak","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Mackenzie","middleName":"","lastName":"Kwak","suffix":""},{"id":607578928,"identity":"3d4aa442-0c7c-48ac-abb4-774da7c6a910","order_by":18,"name":"Yurie Taya","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Yurie","middleName":"","lastName":"Taya","suffix":""},{"id":607578929,"identity":"f4fd704a-7005-4c83-81ee-f703fcbc4365","order_by":19,"name":"Toshio Tsubota","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Toshio","middleName":"","lastName":"Tsubota","suffix":""}],"badges":[],"createdAt":"2026-03-06 04:30:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9045918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9045918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104858485,"identity":"b1cab50c-554d-4827-8749-d114b9b5e03c","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":272687,"visible":true,"origin":"","legend":"\u003cp\u003eStudy area. Left panel) In the map of Japan, Hokkaido island is shaded in grey and enlarged in the middle panel, and Ehime prefecture, where Toyo virus (TOYV) was first identified in ticks, is marked with a black star. Middle panel) Four regions of Hokkaido are highlighted in different colors, and the administrative boundaries of Sapporo and Asahikawa cities are indicated. Locations where TOYV-positive ticks were collected are shown by red (ID:18THF) and black (ID:15THM) dots. Right panel) In the map of the Shiretoko Peninsula, the borders of two towns (Shari and Rausu) are indicated by dashed lines. The Shiretoko National Park is highlighted in green. A human-dominated landscape is shown in dark grey. Elevation is represented using a color gradient from light blue (low elevation) to dark red (high elevation). This map was created by processing raster elevation data from the Geospatial Information Authority of Japan (Global Map Japan version 1.0 Raster Data, released in 2000; accessed in November 2022) and land cover data from the Japan Aerospace Exploration Agency (High-Resolution Land-Use and Land-Cover Map of Japan for 2022, Released in December 2023, Version 23.12; accessed in February 2024). The data are available online at https://www.gsi.go.jp/kankyochiri/gm_japan_e.html and https://www.eorc.jaxa.jp/ALOS/en/dataset/lulc/lulc_v2312_e.htm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/1f0127a3b311a409e0a08996.png"},{"id":104858488,"identity":"a549263d-577c-401e-840b-8bb3da5a33e5","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":253597,"visible":true,"origin":"","legend":"\u003cp\u003eTOYV isolation and characterization. (A) TOYV quantification in brown bears using RT-qPCR. The x-axis shows bear IDs, and the y-axis represents the number of viral RNA copies per 100 ng of total RNA from tissue or plasma. Samples with Ct values \u0026gt;42 are marked as ND. X, not available. (B) RT-qPCR measurement of TOYV in 4 w.o. AG129 infected with brown bear tissue homogenate samples. Mice were sacrificed at 21 days-post infection (dpi). The x-axis represents the brown bear IDs, the sample of which was used for infection. The y-axis is the number of virus RNA copies per 100 ng of total tissue/serum RNA. A red triangle indicates a sample used for virus isolation. (C) Cytopathic effects (CPE) induced by TOYV in Hep3B cells at 6 dpi, compared with the negative control (Mock), and (D) immunofluorescence assay staining TOYV antigen in Hep3B cells at 4 dpi using immunized AG129 mouse serum as a primary antibody and anti-mouse Alexa Fluor 488–labeled IgG as a secondary antibody. (E) TOYV RNA copy number (left panel) and infectious virus titer (right panel) in the supernatant of Hep3B cells following infection at an MOI of 0.001. TCID\u003csub\u003e50\u003c/sub\u003e, 50% tissue culture infectious dose. (F) Transmission electron microscopy image of TOYV particles in the supernatant of infected Hep3B cells. The area outlined by the white rectangle is enlarged on the right, with diameter measurements indicated. White scale bar = 200 nm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/923ba8cef396a75bbf31288d.png"},{"id":104858490,"identity":"1ba7a9d6-ee31-4a3e-ac6d-d8cc4786fc7a","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134551,"visible":true,"origin":"","legend":"\u003cp\u003eTOYV neuroinvasive potential investigation. A) Outline of experiments. A 4-week-old (w.o.) AG129 was intraperitoneally (IP) infected with spleen, liver, and plasma mix of Toyo virus (TOYV) RT-PCR-positive bear (bear ID: 23B51) and sacrificed at 21 days post-infection (dpi). Mouse brain tissue homogenate was then intracerebrally (IC) inoculated into nine suckling 2-day-old (d.o.) AG129 mice. Period of neurological signs observation in 2 d.o. mice is highlighted in grey, days at which pups were sacrificed are indicated with triangles and the number of sacrificed pups indicated underneath the triangles. Virus isolate (23B51b) was inoculated into Neuro-2a mouse neuroblastoma cell line and intraperitoneally (IP) inoculated into three (n=3) adult AG129 and four (n=4) ICR. All mice were sacrificed at 20 dpi and brain and spleen tissues collected. B) RT-qPCR quantification of TOYV in 2 d.o. AG129 brain homogenate. The y-axis shows the virus copies per 10 mg of homogenized brain tissue suspension. Virus copies number of the sample used for pups’ infection is shown as “injected”. For three pups sacrificed on 7 dpi and five sacrificed between 11 and 13 dpi, the mean value with SD is indicated. C) TOYV RNA copy numbers following infection with TOYV isolate 23B51b at an MOI of 0.01 in Neuro-2a cells. D) Immunofluorescence assay (IFA) staining of infected and non-infected (mock) Neuro-2a cells at 8dpi. White scale bar = 50 µm. E) RT-qPCR quantification of TOYV RNA in AG129 and ICR mice spleen and brain tissue. Y-axis indicates viral copy numbers per 100 ng of total tissue RNA and the x-axis indicates individual mouse IDs. ND, not detected.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/50ae9f388a13cfb9cb7c04cc.png"},{"id":104858491,"identity":"911efcd5-a7b9-43e5-ae77-2eb743ab0407","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":254108,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of TOYV gene sequences. Maximum-likelihood phylogenetic trees of viruses within the genus \u003cem\u003eUukuvirus\u003c/em\u003e(A–D) and Toyo viruses (E–H) were constructed using nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) (A, E), glycoprotein precursor (Gn and Gc) (B, F), nucleocapsid protein (N) (C, G), and nonstructural protein (NSs) coding regions (D, H) using IQ-TREE 2. Branch support was evaluated using 10,000 replicates of SH-aLRT and ultrafast bootstrap (UFboot) tests, with nodes showing SH-aLRT support ≥85% (blue circles) and UFboot support ≥95% (orange circles) indicated. Gouleako virus (A, B) or Uukuniemi virus and Silverwater virus (E–H) were used as outgroups but are not shown due to their large genetic divergence. Sequences derived from brown bears are highlighted in red, and sequences from tick pools are shown in green.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/8b32542ffcd9f08e2415b0f5.png"},{"id":105034050,"identity":"67ff4c64-7f71-4b25-9ec6-8d07aac62c1b","added_by":"auto","created_at":"2026-03-20 07:22:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91689,"visible":true,"origin":"","legend":"\u003cp\u003eTOYV antibody screening in wildlife. Brown bears (A), sika deer (B), and raccoons (C) tested for TOYV antibodies by ELISA according to the month and site of capture. Colors indicate the results of the IFA, where “Negative” also includes samples that were considered negative in ELISA and, therefore, not subjected to IFA testing. NP, National Park.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/34ad16bbb35da9cd7450087e.png"},{"id":105036547,"identity":"d6b68a06-f2fb-4203-b4e9-04dc4b0760a4","added_by":"auto","created_at":"2026-03-20 07:34:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/2fd9e12a-a166-4b98-9dd2-e07e54eefcaa.pdf"},{"id":104858487,"identity":"7b08a5f3-ce02-4033-be73-ec8405f6445a","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":208690,"visible":true,"origin":"","legend":"Supplementary Files","description":"","filename":"20260306SupplementaryFiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/3254e16ad9f9d6287f2cf544.pdf"},{"id":104858486,"identity":"0b32917d-3e07-4df8-a26c-3993e58c1c2a","added_by":"auto","created_at":"2026-03-18 04:34:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19835,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9045918/v1/e6ab6e12a1596fd1ed439561.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Circulation of an emerging neurotropic tick-borne phenuivirus in brown bears and wildlife in northern Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTick-borne viruses are important pathogens of public and animal health concern, as most of them are zoonotic, meaning capable of transmitting between species [1]. Wild animals and birds can transport ticks over long distances and serve as reservoirs for tick-borne pathogens, facilitating their spread [2].\u0026nbsp;Habitat\u0026nbsp;loss\u0026nbsp;in wildlife populations, together with\u0026nbsp;human population\u0026nbsp;growth\u0026nbsp;and urbanization,\u0026nbsp;has led to increased\u0026nbsp;contact between humans and wildlife, and\u0026nbsp;consequently\u0026nbsp;with\u0026nbsp;wildlife-associated\u0026nbsp;pathogens\u0026nbsp;[1,3]. This highlights the importance of wildlife surveillance for identifying infections of emerging tick-borne pathogens that may threaten public health currently and\u0026nbsp;in the\u0026nbsp;near future.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe family \u003cem\u003ePhenuiviridae\u0026nbsp;\u003c/em\u003econtains multiple zoonotic viruses, such as severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV), which causes an emerging disease endemic in East Asia as well as in Japan [4]. In ticks, several novel viruses belonging to\u0026nbsp;the family \u003cem\u003ePhenuiviridae\u003c/em\u003e have been detected worldwide [5,6,7,8], including\u0026nbsp;Japan [9,10,11,12,13]. Some of these emerging tick-borne phenuiviruses have been shown to infect\u0026nbsp;humans and animals mostly by serological assays [14,15]. However, for many other identified viruses in this family, the ability to infect mammals remains unclear. For example, Toyo virus (TOYV; genus \u003cem\u003eUukuvirus\u003c/em\u003e) was first identified in \u003cem\u003eHaemaphysalis\u0026nbsp;\u003c/em\u003e\u003cem\u003eformosensis\u003c/em\u003e ticks collected in southern Japan [13] (Fig. 1), while infection in mammals\u0026nbsp;and\u0026nbsp;distribution in other regions have\u0026nbsp;not been reported. Because most epidemiological studies of tick-borne phenuiviruses in animals, including SFTSV, have relied on tick screening and antibody testing of animals, direct detection of viral genomes in wildlife is necessary to assess their current prevalence and host diversity.\u003c/p\u003e\n\u003cp\u003eIn Hokkaido, the northern island of Japan, land use changes and the expansion of agricultural lands in the past, followed by their abandonment, have created suitable habitats for some wild animal species [16]. Together with a lack of buffer zones between cities and forests, this has led to an increase in intrusions of wild animals such as feral raccoons (\u003cem\u003eProcyon lotor\u003c/em\u003e), Hokkaido sika deer (\u003cem\u003eCervus nippon yesoensis\u003c/em\u003e), and Hokkaido brown bears (\u003cem\u003eUrsus arctos yesoensis\u003c/em\u003e) in urban and suburban areas [17], potentially facilitating the spread of tick-borne diseases.\u0026nbsp;Although rodents, birds, small mammals, and some ungulates have been relatively well studied as hosts of tick-borne pathogens in Hokkaido\u0026nbsp;[18,19,20,21], data on medium- to large-sized animals remain limited. Bears, in particular, may play an important role in spreading tick-borne pathogens due to their\u0026nbsp;large habitat area\u0026nbsp;and long lifespans [22], but no studies have assessed the brown bear exposure to tick-borne viruses in Hokkaido.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we conducted genetic surveillance for phenuiviruses in brown bears, raccoons, and sika deer across Hokkaido, focusing on animals that intrude into human settlements. Interestingly, infections with an emerging tick-borne virus, i.e. TOYV, were identified in multiple brown bears. This represents the first report of TOYV infection in mammals, and the successful isolation of TOYV may constitute the first isolation of a tick-borne virus from ursids. We demonstrate that TOYV exhibits neuroinvasive and neurotropic potential both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, genetic screening of bears and ticks, together with serological surveys of wildlife, revealed endemic circulation of TOYV in Hokkaido. These findings raise concerns of TOYV zoonotic potential and its impact on animal and human health. Overall, this study highlights the importance of systematic surveillance at the wildlife\u0026ndash;human interface, targeting diverse animal species to identify and monitor emerging tick-borne viruses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIdentification of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTOYV\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;infection in\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ewildlife\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween 2022 and 2023, lymph node or spleen samples from raccoons and sika deer captured in Sapporo and Asahikawa, and spleen samples from brown bear captured in western (Shari town) and eastern (Rausu town) sides of the Shiretoko Peninsula (Figure 1) were tested by RT-PCR targeting multiple tick-borne viruses in the family \u003cem\u003ePhenuiviridae\u003c/em\u003e. Nine of 149 tested bears captured in 2023 were positive, while all raccoons and sika deer were negative (Table 1). Seven sequences of the RT-PCR fragments shared 97.7% nucleotide similarity with the TOYV RNA-dependent RNA polymerase (RdRp) gene, and two partial sequences with a high number of ambiguities showed 88.0% and 89.2% similarity (Supplementary Table 4).\u003c/p\u003e\n\u003cp id=\"_Toc214898375\"\u003e\u003cstrong\u003e\u003cem\u003eTOYV isolation and characterization\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp id=\"_Toc214898377\"\u003eThe amounts of TOYV RNA in PCR-positive bear tissues were quantified. Most bears had detectable viral RNA in the liver and spleen, and a few in plasma and lung samples (Figure 2A). For isolation of TOYV, bear samples with high virus load were administered intraperitoneally to adult AG129 mice. While no weight loss or clinical signs were observed, viral RNA was detected in\u0026nbsp;tissues at 21 dpi, with the highest quantities in spleen and brain (Figure 2B). A brain homogenate sample with the highest viral load from the mouse (infected with samples of bear ID: 23B51) was then inoculated into mammalian cell lines for the \u003cem\u003ein vitro\u003c/em\u003e isolation. No\u0026nbsp;CPE or continuous viral growth\u0026nbsp;could be observed in Vero E6, BHK21, and Huh-7 cells (data not shown). Productive\u0026nbsp;viral growth resulting in cell death was detected only in Hep3B cells (Figure 2C), and viral antigen expression was confirmed by IFA using immunized mouse serum as primary and anti-mouse IgG as secondary antibodies (Figure 2D).\u0026nbsp;The TOYV isolate (named 23B51b) replicated efficiently in Hep3B (Figure 2E).\u0026nbsp;Electron microscopy of the supernatant of infected Hep3B cells revealed spherical particles of approximately 90 nm, matching the\u0026nbsp;morphology of viruses in the genus \u003cem\u003eUukuvirus\u003c/em\u003e (Figure 2F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNeuroinvasive potential and neurotropism of TOYV\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the replication of TOYV in the brain, nine newborn AG129 mice were intracerebrally inoculated with the brain sample used for virus isolation (Figure 3A). Neurological signs, including ataxia, delayed righting reflex, and impaired movements, developed between 11 and 13 dpi and were accompanied by a marked increase in viral RNA levels in brain tissue (Figure 3B). The neurotropism of the TOYV isolate in neural tissue was further assessed using the mouse neuroblastoma cell line Neuro-2a (Figure 3A). Viral RNA levels increased over time without showing CPE (Figure 3C), with virus titer reaching 2x10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e/ml at 8 dpi. Viral antigen was also detected in infected cells by IFA (Figure 3D). The neuroinvasive potential of TOYV was then assessed by intraperitoneal inoculation of the TOYV isolate 23B51b into adult AG129 and ICR mice (Figure 3A). Although all inoculated AG129 and ICR mice showed detectable viral loads in their spleens, virus in the brain was detected in only one AG129 mouse (Figure 3E).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eTOYV screening in ticks\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo determine a potential vector species of ticks for TOYV, ticks collected on the Shiretoko Peninsula in 2024 and 2025 were pooled by species and developmental stage into a total of 102 pools and screened for TOYV using RT-qPCR, with all samples testing negative (Supplementary Table 5). A retrospective analysis of archived tick metagenomes was performed, detecting TOYV genomes in a pool of \u003cem\u003eHaemaphysalis megaspinosa\u003c/em\u003e nymphs collected in April 2015 and in a pool of \u003cem\u003eHaemaphysalis flava\u003c/em\u003e females collected in October 2018 from central Hokkaido (previously reported in GenBank as LC604128).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTOYV genome sequencing and phylogenetic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCoding sequences of the RdRp, the glycoprotein precursor (Gn and Gc), and nucleocapsid (N) and nonstructural (NSs) proteins encoded by the L, M, and S segment RNAs, respectively, were obtained from tissue samples of six of the RT-PCR\u0026ndash;positive brown bears by using primers designed for the TOYV reference. Complete sequences of coding regions of TOYV in the archived \u003cem\u003eH. flava\u003c/em\u003e pool (18THF) were also obtained by metagenome sequencing. Phylogenetic analyses showed that all available TOYV sequences across three genomic segments were closely related and clustered into a highly supported clade with the corresponding TOYV sequences available in GenBank, which were obtained from \u003cem\u003eH. formosensis\u003c/em\u003e ticks collected in Ehime Prefecture, Japan (Figure 4A-D). The TOYV cluster was included in the Kaisodi group, which is consistent with previous reports. Additional analysis within the TOYV sequences group, including a partial sequence obtained by a metagenome sequencing of a \u003cem\u003eH. megaspinosa\u003c/em\u003e tick pool (15THM), showed that TOYVs from bears 23B53 and 23B73 clustered together with 15THM, while sequences from bears 23B51, 23B55, 23B65, and 23B72 formed a separate cluster (Figure 4E-F).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eTOYV screening in wildlife\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;in Hokkaido\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFirst, to examine the infections of TOYV among bears, bear tissues collected in 2024 were subjected to RT-PCR, and those collected in 2025 to RT-qPCR for genetic screening. None of the 19 bears in 2024 tested positive, while\u0026nbsp;six of 60\u0026nbsp;animals were positive in 2025 (Supplementary Table 6).\u003c/p\u003e\n\u003cp\u003eTo further assess TOYV exposure in brown bears and other wildlife species in Hokkaido, serum samples from 208 brown bears, 161 sika deer, 210 raccoons, and 187 rodents were subjected to serological screening using ELISA, with positive results confirmed by IFA (Table 2). Among brown bears, seven out of 77 (9.1%) in Shari and two out of 50 (4%) in Rausu were seropositive in 2023. In total, ten out of 64 in 2025 tested positive, while 17 captured in 2024 were negative. All ELISA-positive bears in 2023 and 2025, as well as all PCR-positive bears in 2025, were captured in autumn, 22 of 28 (78.6%) being females (Table 3) among 126 females out of 208 captured bears (60.6%). Notably, a few cubs that were born in 2025 were found infected (Table 3). A seasonal difference in sampling was observed, with a larger number of animals tested in autumn (Figure 5A). Among sika deer, none tested positive in Shiretoko National Park or northern Hokkaido (Asahikawa), whereas a few captured in central Hokkaido (Sapporo) were seropositive (Table 2). However, sample sizes varied by region and season (Figure 5B). While none of the raccoons captured in Asahikawa were positive, seroprevalence in Sapporo consistently exceeded 25% between 2022 and 2024, with positive animals detected from spring through autumn (Table 2; Figure 5C). All tested rodents captured in central Hokkaido were seronegative (Table 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we performed genetic screening of tick-borne phenuiviruses in wildlife in Hokkaido, Japan, and identified brown bear infections with TOYV, a phenuivirus previously reported in ticks in Japan [13]. TOYV was successfully isolated, likely representing the first recovery of a tick-borne virus from any ursid species in the world, as studies of viral infections in ursids remain limited [23,24]. Moreover, our findings imply the neuroinvasive and neurotropic potential of TOYV actively circulating within Japan, which may pose risks to impact animal and human health.\u003c/p\u003e\n\u003cp\u003eWe successfully isolated TOYV from the brain tissue of mice inoculated with bear samples intraperitoneally, suggesting that the virus has neuroinvasive potential. This was further supported by the observations \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, particularly with neurological signs and active viral replication in the brain tissue of infected newborn mice and the detection of viral RNA in the brains of adult AG129 mice following intraperitoneal infection with the virus isolate. Since virus detection in the mouse brain was observed only in interferon-receptor knockout AG129, but not in inbred wild-type ICR mice, the host innate immune response plays an important role in protecting the central nervous system from TOYV infection, as reported for other neurotropic viruses [25,26]. This study may represent the first report of neuroinvasive potential among uukuviruses, including Kaisodi group phenuiviruses, to which TOYV belongs, which were shown to infect mammals and birds [27-29]. Neurological diseases, as well as virus detection in cerebrospinal fluid, were observed in patients infected with human pathogenic tick-borne phenuiviruses, such as SFTSV and Lone Star virus [30,31], raising the possibility that neurotropism may extend to multiple members of the \u003cem\u003ePhenuiviridae\u003c/em\u003e family and underscoring the need for further investigation of TOYV neurovirulence.\u003c/p\u003e\n\u003cp\u003eOur wildlife surveillance revealed infections of TOYV in multiple species of mammals as well as ticks in Hokkaido, distant from the place where TOYV was initially identified in ticks [13]. Hokkaido is located on the East Asian\u0026ndash;Australasian Flyway, and the Shiretoko Peninsula serves as a stopover for multiple migratory bird species that can introduce ticks from other regions of Japan or overseas [18,32].\u0026nbsp;Thus, TOYV may have been introduced to Hokkaido or vice versa via migratory birds.\u0026nbsp;TOYV derived from brown bears and ticks phylogenetically showed no clear geographic clustering by sampling location, suggesting viral circulation within Hokkaido and also Japan, likely facilitated by long-distance transportation of ticks by birds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTOYV-infected brown bears were predominantly female and captured on the western side of the Shiretoko Peninsula (Shari) during autumn. Since female bears\u0026rsquo; typical home ranges are less than 30 km\u0026sup2;, much smaller than those of male bears [33], TOYV infections likely occurred within limited areas of the Shiretoko Peninsula. The geographical distribution of TOYV in bears may therefore be influenced by the locations of migratory birds\u0026apos; stopover sites, as well as by differences in tick species composition between the two coasts of the peninsula, which are separated by a mountain range that shapes the regional climate [34]. TOYV\u0026nbsp;was initially detected in \u003cem\u003eH.\u0026nbsp;\u003c/em\u003e\u003cem\u003eformosensis\u003c/em\u003e in southern Japan [13] and\u0026nbsp;retrospectively detected in\u0026nbsp;\u003cem\u003eH.\u003c/em\u003e\u003cem\u003e\u0026nbsp;megaspinosa\u003c/em\u003e ticks and \u003cem\u003eH. flava\u003c/em\u003e ticks collected in the central Hokkaido long before the detection of infection in brown bears in Shiretoko. Viruses in the Kaisodi group are known to be transmitted by \u003cem\u003eHaemaphysalis\u003c/em\u003e ticks\u0026nbsp;[29], and\u0026nbsp;our findings strongly suggest the involvement of \u003cem\u003eHaemaphysalis\u003c/em\u003e ticks in TOYV circulation. We also revealed that the seroprevalence of TOYV in raccoons and sika deer was different among regions in Hokkaido, and similar to the observation of the TOYV-positive bears, the infected animals were predominantly found in autumn. These regional and seasonal occurrences of TOYV infections may be determined by the abundance and seasonality of \u003cem\u003eHaemaphysalis\u003c/em\u003e ticks, as well as regional and seasonal differences in the host preference of ticks [35,36]. Further understanding of the vector and host competencies of each tick and animal species for TOYV will help to reveal the mechanism behind the emergence of tick-borne phenuiviruses.\u003c/p\u003e\n\u003cp\u003eIn summary, we report the infections of the emerging phenuivirus, potentially pathogenic to animals and humans, in wildlife in Hokkaido, Japan, and demonstrate the TOYV active circulation in the region. This underscores the need for further investigation of TOYV pathogenicity and continued monitoring to assess its spread in animals and potentially humans. Due to the limited availability of wildlife samples, we could not examine TOYV infections in the brains of wildlife in the present study, and thus, the impact of TOYV spread on wildlife and public health should be carefully investigated. This study emphasizes that wild animals are important targets for monitoring the emergence of novel tick-borne viral infections and supports the integration of systematic wildlife pathogen screening into national health programs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample collection from wildlife\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples of sika deer and raccoons from central (Sapporo) and northern (Asahikawa) Hokkaido, as well as serum samples of live-captured Hokkaido sika deer within Shiretoko National Park, were collected as part of the previous study [37]. Brown bears outside the Shiretoko National Park are culled annually for management purposes and used for sampling on both the eastern and western coasts, which are separated by a mountain range (Figure 1). Spleen, liver, lung, and blood samples were collected from the brown bears between March 2023 and October 2025. Samples were stored at \u0026minus;20\u0026deg;C prior to delivery to Hokkaido University for further processing. The brown bear age was estimated based on body size, observation of canine tooth development, and the presence of the mother at the time of capture, and is reported as \u0026lt;1 year for dependent cubs assumed to have been born in the year of capture [38].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRodent trapping was performed in Central Hokkaido (Figure 1) during May 2025 to June 2025 using Sherman traps, and captured rodents were euthanized under isoflurane anesthesia, followed by cervical dislocation. Blood was collected directly from the heart and centrifuged to separate serum, followed by storage at \u0026ndash;20\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA extraction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA of animal spleen and lymph node tissue samples was extracted using NucleoSpin RNA Kit (TAKARA Bio Inc.). Briefly, tissues initially stored at \u0026minus;20 \u0026deg;C were cut into smaller pieces and re-frozen at \u0026minus;80 \u0026deg;C prior to RNA extraction. For extraction, lysis buffer supplemented with carrier RNA was added directly to the frozen tissue, and samples were homogenized at 3,000 rpm for 1 min at 4\u0026deg;C. The lysates were then filtered through a NucleoSpin Filter, and RNA extraction was performed according to the manufacturer\u0026rsquo;s instructions. The RNA was eluted in 50-60 \u0026micro;L of RNase-free water and stored at \u0026ndash;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-PCR and Sanger sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-step RT-PCR was performed with primers targeting the conserved region of the genome among multiple viruses in \u003cem\u003ePhenuiviridae\u003c/em\u003e family, as described previously [12]. Amplified partial genomes were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions in the 3500xL Genetic Analyzer (Applied Biosystems). Obtained sequences were compared to the sequences in the NCBI database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers and a tag-labeled probe targeting TOYV S segment were designed using the IDT PrimerQuest Tool (Integrated DNA Technologies) (Supplementary Table 1). The assay was performed with One Step PrimeScript III RT-qPCR Mix (TAKARA Bio Inc.). A standard curve was generated using 10-fold serial dilutions of DNA amplified with the same primers, with a detection limit of 40 copies/well. The thermal profile included 25\u0026deg;C for 10 min, 52\u0026deg;C for 2 min, followed by 45 cycles of 95\u0026deg;C for 10 sec and 60\u0026deg;C for 30 sec, and was performed on a qTOWER3 Real-time PCR Thermal Cycler (Analytik Jena AG). All the samples were measured in duplicate, and samples with the cycle threshold (Ct) less than 42 in at least one replicate were considered positive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMouse infection experiments for in vivo v\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eirus isolation and characterization\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType I and II interferon receptor-knockout (AG129) mice were in-house bred (obtained from Marshall BioResources), and\u0026nbsp;ICR inbred mice were obtained from Japan SLC. Experimental design for animal experimentation is indicated in Figure 3A.\u003c/p\u003e\n\u003cp\u003eFor virus isolation using mice, samples (i.e., plasma and/or tissue homogenates) from RT-PCR-positive brown bears were inoculated intraperitoneally into adult AG129 mice. Animals were monitored daily, and internal organs were collected for RNA quantification with RT-qPCR. The brain sample from an AG129 mouse with the highest viral load was used for further passaging and for intracerebral inoculation into a litter of 2-day-old (d.o.) AG129 pups. Three asymptomatic pups inoculated with the adult AG129 brain sample were euthanized at 7 dpi, and one at 21 dpi. Five pups that developed neurological signs were euthanized at 11\u0026ndash;13 dpi. The brain tissues of pups were collected for viral RNA quantification.\u003c/p\u003e\n\u003cp\u003eFor examining the pathogenesis of TOYV infections, especially neurological signs, four adult ICR and three adult AG129 were intraperitoneally inoculated with 3,000 TCID\u003csub\u003e50\u003c/sub\u003e of TOYV isolate. Animals were monitored daily and euthanized at 20 dpi. Brain and spleen tissues were collected for viral RNA quantification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVero E6 (African green monkey kidney), BHK21 (Syrian hamster kidney), Huh-7 (human hepatoma), and Hep3B (human liver carcinoma) cells were cultured in Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (DMEM) (Nacalai Tesque Inc.) supplemented with 10% Fetal Bovine Serum (FBS) (ICN Biomedicals Inc.), 1% Penicillin\u0026ndash;Streptomycin (Thermo Fisher Scientific), and 1% L-glutamine (FUJIFILM Wako Chemicals). Neuro-2a (mouse neuroblastoma) cells were cultured in Eagle\u0026apos;s Minimum Essential Medium (EMEM) (FUJIFILM Wako Chemicals) supplemented with 1% MEM Non-Essential Amino Acids Solution (NEAA) (Thermo Fisher Scientific), 10% FBS, and 1% Penicillin\u0026ndash;Streptomycin. Following infection, all cells were maintained in medium containing 2% FBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVirus isolation in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAG129 mouse brain tissue was homogenized in DMEM supplemented with 10% FBS and centrifuged. The supernatant was inoculated onto VeroE6, BHK21, Huh-7, and Hep3B cell monolayers in T25 culture flasks. After 1 h of incubation at 37 \u0026deg;C, cells were washed with PBS and overlaid with DMEM containing 2% FBS. Maximum three passages (4\u0026ndash;7 days each) were performed. Cells were monitored for cytopathic effects (CPE), and viral RNA in the supernatant was quantified by TOYV-specific RT-qPCR. Infection was confirmed by immunofluorescence assay using mouse convalescent serum as the primary antibody.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransmission electron microscopy (TEM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupernatant from Hep3B cells infected with TOYV was centrifuged at 3,500 rpm for 10 min at 4\u0026deg;C. The virus was then pelleted down through a 20% sucrose cushion by ultracentrifugation at 28,000 rpm for 2 h at 4\u0026deg;C using an SW32Ti rotor in an Optima L-90K centrifuge (Beckman Coulter). The resulting pellet was resuspended in 100 \u0026micro;L of PBS and stored overnight at 4\u0026deg;C. Concentrated virions were placed onto carbon-coated, ion-sputtered grids (Nisshin EM) for 1 min and negatively stained with 2% phosphotungstic acid (PTA) (pH 7.0) for 15 s. Transmission electron microscopy was performed using a Hitachi H-7650 instrument (Hitachi High-Tech).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHep3B cells infected with TOYV were incubated with lysis buffer at 4\u0026deg;C for 30 min (ATTO co., ltd). The samples were centrifuged at 15,000 rpm for 15 min at 4\u0026deg;C, supernatants collected and stored at \u0026ndash;80\u0026deg;C till use. The lysate of cells infected with TOYV was used as ELISA antigen, with uninfected cell lysate as a negative control antigen. Lysates were diluted in PBS, coated onto plates, and incubated overnight at 4\u0026deg;C. After a single tris-buffered saline with Tween 20 (TBS-T) wash, 50 \u0026micro;L of plasma or serum (1:400 diluted in Blocking One (Nacalai Tesque Inc.)) was added to each well and incubated for 1 h at room temperature. Following three washes, HRP-conjugated Protein A/G (32490, Thermo Fisher Scientific) (for sika deer, raccoon, and brown bear samples) or HRP-conjugated anti-mouse antibodies (ab6789, Abcam) (for rodent samples) were applied, followed by incubation at room temperature for 1 h. Plates were washed again, developed with 100 \u0026micro;L 3,3\u0026apos;,5,5\u0026apos;-tetramethylbenzidine (TMB) substrate for 15-20 min at room temperature, and the reaction was stopped with 100 \u0026micro;L of 1M HCl. Optical density (OD) was measured at 450 nm in a plate reader (Multiskan FC). OD difference (∆OD) between the antigen-coated well and the negative control cell lysate-coated well was calculated, and the cutoff value was measured according to the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"269\" height=\"21\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eNegative samples were defined as brown bear sera samples collected in 2023 that tested negative by immunofluorescent assay (IFA, see below) at a 1:50 dilution. Based on this calculation, the cutoff value was set at 0.3 (all ∆OD are provided in Supplementary Figure 1). Notably, for raccoon sera, several samples with ∆OD between 0.3 and 0.5 were found to be IFA-negative. Therefore, to maximize diagnostic specificity and reduce the risk of false-positives, the cutoff for raccoons was retrospectively increased to 0.5 (a breakdown of ∆OD for raccoons based on IFA results is provided in Supplementary Figure 2). Sera samples tested as positive in ELISA were examined by IFA as described below.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIFA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e96-well flat-bottom plates were coated with e-poly-L-lysine (1:10 in PBS), incubated at 37\u0026deg;C for 30 min. Hep3B cells were then seeded into the plates, grown to monolayers, and infected with 10 TCID\u003csub\u003e50\u003c/sub\u003e (50% tissue culture infectious dose) of TOYV isolate. After 4 days of culture, cells were fixed with 4% paraformaldehyde (PFA) (FUJIFILM Wako Chemicals) for 1 h at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 (SIGMA-ALDRICH) for 15 min, then washed again.\u003c/p\u003e\n\u003cp\u003eAnimal sera or plasma samples were heat-inactivated at 56\u0026deg;C for 30 min and diluted 1:50 in culture medium. 50 \u0026micro;L of diluted sample was added per well and incubated for 1 h at room temperature. After three PBS washes, cells were incubated with FITC-conjugated Protein A/G (BioVision), then nuclei were counterstained with DAPI (FUJIFILM Wako Chemicals) and images taken with a fluorescence microscope (ZEISS). For mouse sera, Alexa Fluor 488\u0026ndash;labeled anti-mouse IgG (A32723 or A-11029, Invitrogen, Thermo Fisher Scientific), and for raccoons, with high background staining, anti-raccoon HRP-conjugated IgG (A140-123P, Bethyl Laboratories) were used as the secondary antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTOYV survey in ticks\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 2024 and 2025, ticks were collected on the Shiretoko Peninsula using flannel flags during spring-early summer (April, May, and June) and autumn (September and October). Specimens were\u0026nbsp;identified morphologically [39], and\u0026nbsp;total DNA and RNA were extracted from individual ticks or tick pools (up to 10 individuals of the same sex and developmental stage) using the BlackPrep Tick DNA/RNA kit (Analytik Jena) following\u0026nbsp;the manufacturer\u0026rsquo;s protocol. Tick RNA was screened for TOYV using RT-qPCR as described above. In addition, a retrospective analysis of TOYV was performed using a BLASTn search of assembled sequences from in-house metagenomic RNA-seq data derived from ticks collected in Hokkaido [40], as well as a BLASTn search of the GenBank database. Additional sequencing and viral genome assembly were performed as described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTOYV genome sequencing and phylogenetic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor determining the full-genome sequences of TOYVs from brown bears, the RT-PCR positive bear spleen and liver RNAs were used for cDNA synthesis with the PrimeScript II 1\u003csup\u003est\u003c/sup\u003e Strand cDNA Synthesis Kit (TAKARA Bio Inc) following the manufacturer\u0026rsquo;s instructions. The cDNA was used as a template for PCR amplification of TOYV genome fragments with primers designed based on TOYV reference sequences in GenBank (Accession numbers: LC618931, LC618932, LC618933). Amplified DNA was sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit with additional customized primers. To assemble TOYV genomes from two TOYV-positive tick pools determined during the retrospective virus genome screening, paired-end Illumina NextSeq reads were quality-checked and trimmed to remove adapter sequences and low-quality bases. Filtered reads were then mapped to a reference TOYV genome retrieved from GenBank using standard alignment tools in Geneious Prime software version 2025.2.2 (https://www.geneious.com), and consensus sequences were generated from the mapped reads. All TOYV sequences obtained in this study are deposited in the GenBank database (their accession numbers are listed in the Supplementary Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe obtained sequences were aligned with the corresponding TOYV genome and representative members of the family \u003cem\u003ePhenuiviridae\u003c/em\u003e (accession numbers provided in Supplementary Table 3). \u0026nbsp;Multiple sequence alignment of the nucleotide sequence for four protein coding genes (RdRp, G, N and NSs) was performed using the Translation Align function in the Geneious Prime software. Alignment curation was performed manually by removing gap-containing regions while preserving the codon structure of the coding sequences. Maximum-likelihood phylogenetic trees for four genes were constructed with IQ-TREE 2 [41] with nucleotide substitution models selected by ModelFinder [42] under the -m MFP option based on partitioned codon positions. Node support was assessed using 10,000 replicates of the Shimodaira\u0026ndash;Hasegawa-like approximate likelihood ratio test (SH-aLRT) [43] and ultrafast bootstrap approximation (UFboot) [44]. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The study was approved by the Animal Care and Use Committee of Hokkaido University under permits No. 22-0016, No. 18-0001, No. 18-0083, No. 23-0014, No. 23-0178, and No. 25-0119. Animal infection experiments were carried out in the biosafety level 2 facility at the Hokkaido University International Institute for Zoonosis Control. Permission for rodent capture for academic purposes was obtained from the Hokkaido Government.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the Shiretoko Nature Foundation staff and members of the Wildlife Biology and Medicine Laboratory at Hokkaido University for their assistance in the sample collection. We appreciate the members of the Division of Risk Analysis and Management at the International Institute for Zoonosis Control, especially Asako Aoshima/Shigeno, for the technical assistance. We also appreciate Soyo Ohsako, Yuki Shirota, Kenya Suzuki, Yuta Tsukahara, and Natsuki Miyazaki from the Faculty of Veterinary Medicine at Hokkaido University for their contribution to tick collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.K. – study design and implementation, sample acquisition, data analysis, funding acquisition, and manuscript preparation. M.S. – management, funding acquisition, and sample acquisition. Y.O. – data analysis and visualization. M.I. – sample and funding acquisition. J.L. and Y.M. – study implementation. Kh.S., T.K., M.Y., Ko.S., and L.U. – sample acquisition. Y.O., M.S., N.K., E.F., R.N., M.L.K., and Y.T. – sample acquisition and expertise sharing. T.T. – study design and supervision, funding acquisition. K.M. – study design and implementation, expertise sharing, and funding acquisition. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJones, K. E. et al. Global trends in emerging infectious diseases. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e451\u003c/strong\u003e, 990\u0026ndash;993 (2008).\u003c/li\u003e\n\u003cli\u003eBaneth, G. Tick-borne infections of animals and humans: a common ground. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 591\u0026ndash;596 (2014).\u003c/li\u003e\n\u003cli\u003eHassell, J. M. et al. Urbanization and disease emergence: dynamics at the wildlife-livestock-human interface. \u003cem\u003eTrends Ecol. Evol.\u003c/em\u003e\u003cstrong\u003e32\u003c/strong\u003e, 55\u0026ndash;67 (2017).\u003c/li\u003e\n\u003cli\u003eSilvas, J. A. \u0026amp; Aguilar, P. V. The emergence of severe fever with thrombocytopenia syndrome virus. \u003cem\u003eAm. J. Trop. Med. Hyg.\u003c/em\u003e\u003cstrong\u003e97\u003c/strong\u003e, 992\u0026ndash;996 (2017).\u003c/li\u003e\n\u003cli\u003eLi, C. X. et al. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. \u003cem\u003eeLife\u003c/em\u003e\u003cstrong\u003e4\u003c/strong\u003e, e05378 (2015).\u003c/li\u003e\n\u003cli\u003eBrinkmann, A. et al. A metagenomic survey identifies Tamdy orthonairovirus as well as divergent phlebo-, rhabdo-, chu- and flavi-like viruses in Anatolia, Turkey. \u003cem\u003eTicks Tick Borne Dis.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 1173\u0026ndash;1183 (2018).\u003c/li\u003e\n\u003cli\u003eKoka, H. et al. Detection and prevalence of a novel Bandavirus related to Guertu virus in \u003cem\u003eAmblyomma gemma\u003c/em\u003e ticks and human populations in Isiolo County, Kenya. \u003cem\u003ePLoS One\u003c/em\u003e\u003cstrong\u003e19\u003c/strong\u003e, e0310862 (2024).\u003c/li\u003e\n\u003cli\u003eShen, S. et al. A novel tick-borne phlebovirus, closely related to severe fever with thrombocytopenia syndrome virus and Heartland virus, is a potential pathogen. \u003cem\u003eEmerg. Microbes Infect.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 95 (2018).\u003c/li\u003e\n\u003cli\u003eEjiri, H. et al. Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from \u003cem\u003eHaemaphysalis flava\u003c/em\u003e ticks in Japan. \u003cem\u003eVirus Res.\u003c/em\u003e\u003cstrong\u003e244\u003c/strong\u003e, 252\u0026ndash;261 (2018).\u003c/li\u003e\n\u003cli\u003eMatsuno, K. et al. The unique phylogenetic position of a novel tick-borne phlebovirus ensures an ixodid origin of the genus \u003cem\u003ePhlebovirus\u003c/em\u003e. \u003cem\u003emSphere\u003c/em\u003e\u003cstrong\u003e3\u003c/strong\u003e, e00239-18 (2018).\u003c/li\u003e\n\u003cli\u003eMekata, H., Kobayashi, I. \u0026amp; Okabayashi, T. Detection and phylogenetic analysis of Dabieshan tick virus and Okutama tick virus in ticks collected from Cape Toi, Japan. \u003cem\u003eTicks Tick Borne Dis.\u003c/em\u003e 14, 102237 (2023).\u003c/li\u003e\n\u003cli\u003eMatsuno, K. et al. Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus. \u003cem\u003eJ. Virol.\u003c/em\u003e 89, 594\u0026ndash;604 (2015).\u003c/li\u003e\n\u003cli\u003eKobayashi, D. et al. Toyo virus, a novel member of the Kaisodi group in the genus \u003cem\u003eUukuvirus \u003c/em\u003e(family \u003cem\u003ePhenuiviridae\u003c/em\u003e) found in \u003cem\u003eHaemaphysalis formosensis\u003c/em\u003e ticks in Japan. \u003cem\u003eArch. Virol.\u003c/em\u003e 166, 2751\u0026ndash;2762 (2021).\u003c/li\u003e\n\u003cli\u003eTran, N. T. B. et al. Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan. \u003cem\u003eJ. Vet. Med. Sci.\u003c/em\u003e 84, 82\u0026ndash;89 (2022).\u003c/li\u003e\n\u003cli\u003eTorii, S. et al. Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles. \u003cem\u003eTicks Tick Borne Dis.\u003c/em\u003e 10, 328\u0026ndash;335 (2019).\u003c/li\u003e\n\u003cli\u003eOhashi, H. et al. Land abandonment and changes in snow cover period accelerate range expansions of sika deer. \u003cem\u003eEcol. Evol.\u003c/em\u003e 6, 7763\u0026ndash;7775 (2016).\u003c/li\u003e\n\u003cli\u003eHokkaido Government Environmental Bureau. https://www.pref.hokkaido.lg.jp/ks/skn/ (accessed 3 December 2025).\u003c/li\u003e\n\u003cli\u003eNishino, A. et al. Transboundary movement of Yezo virus via ticks on migratory birds, Japan, 2020\u0026ndash;2021. \u003cem\u003eEmerg. Infect. Dis.\u003c/em\u003e 30, 2674\u0026ndash;2678 (2024).\u003c/li\u003e\n\u003cli\u003eJamsransuren, D. et al. Epidemiological survey of tick-borne encephalitis virus infection in wild animals on Hokkaido and Honshu Islands, Japan. \u003cem\u003eJpn. J. Vet. Res.\u003c/em\u003e 67, 163\u0026ndash;172 (2019).\u003c/li\u003e\n\u003cli\u003eSashika, M. et al. Molecular survey of rickettsial agents in feral raccoons (\u003cem\u003eProcyon lotor\u003c/em\u003e) in Hokkaido, Japan. \u003cem\u003eJpn. J. Infect. Dis.\u003c/em\u003e 63, 353\u0026ndash;354 (2010).\u003c/li\u003e\n\u003cli\u003eKodama, F. et al. A novel nairovirus associated with acute febrile illness in Hokkaido, Japan. \u003cem\u003eNat. Commun.\u003c/em\u003e 12, 5539 (2021).\u003c/li\u003e\n\u003cli\u003eTiffin, H. S., Skvarla, M. J. \u0026amp; Machtinger, E. T. Tick abundance and life-stage segregation on the American black bear (\u003cem\u003eUrsus americanus\u003c/em\u003e). \u003cem\u003eInt. J. Parasitol. Parasites Wildl.\u003c/em\u003e 16, 208\u0026ndash;216 (2021).\u003c/li\u003e\n\u003cli\u003eShi, Y. et al. Extensive cross-species transmission of pathogens and antibiotic resistance genes in mammals neglected by public health surveillance. \u003cem\u003eCell\u003c/em\u003e 188, 6591\u0026ndash;6605.e14 (2025).\u003c/li\u003e\n\u003cli\u003eAlex, C. E. et al. Viruses in unexplained encephalitis cases in American black bears (\u003cem\u003eUrsus americanus\u003c/em\u003e). \u003cem\u003ePLoS One\u003c/em\u003e 15, e0244056 (2020).\u003c/li\u003e\n\u003cli\u003eMilora, K. A. \u0026amp; Rall, G. F. Interferon control of neurotropic viral infections. \u003cem\u003eTrends Immunol.\u003c/em\u003e 40, 842\u0026ndash;856 (2019).\u003c/li\u003e\n\u003cli\u003eChotiwan, N. et al. Type I interferon shapes brain distribution and tropism of tick-borne flavivirus. \u003cem\u003eNat. Commun.\u003c/em\u003e 14, 2007 (2023).\u003c/li\u003e\n\u003cli\u003ePalacios, G. et al. Characterization of the Uukuniemi virus group (\u003cem\u003ePhlebovirus: Bunyaviridae\u003c/em\u003e): evidence for seven distinct species. \u003cem\u003eJ. Virol.\u003c/em\u003e 87, 3187\u0026ndash;3195 (2013).\u003c/li\u003e\n\u003cli\u003eHoff, G. L. et al. Isolations of Silverwater virus from naturally infected snowshoe hares and \u003cem\u003eHaemaphysalis\u003c/em\u003e ticks from Alberta and Wisconsin. \u003cem\u003eAm. J. Trop. Med. Hyg.\u003c/em\u003e 20, 320\u0026ndash;325 (1971).\u003c/li\u003e\n\u003cli\u003eInternational Committee on Taxonomy of Viruses. https://ictv.global/report/chapter/phenuiviridae/phenuiviridae/uukuvirus (accessed 28 January 2026).\u003c/li\u003e\n\u003cli\u003eShan, D. et al. Severe fever with thrombocytopenia syndrome with central nervous system symptom onset: a case report and literature review. \u003cem\u003eBMC neurology\u003c/em\u003e 24, 158 (2024).\u003c/li\u003e\n\u003cli\u003eChiu, CY. et al. Two Human Cases of Fatal Meningoencephalitis Associated with Potosi and Lone Star Virus Infections, United States, 2020-2023. \u003cem\u003eEmerging infectious diseases\u003c/em\u003e 31, 215-221 (2025).\u003c/li\u003e\n\u003cli\u003eNakao, R. et al. Amblyomma testudinarium infestation on a brown bear (\u003cem\u003eUrsus arctos yesoensis\u003c/em\u003e) captured in Hokkaido, a northern island of Japan. \u003cem\u003eParasitol. Int.\u003c/em\u003e 80, 102209 (2021).\u003c/li\u003e\n\u003cli\u003eShimozuru, M. et al. Estimation of breeding population size using DNA-based pedigree reconstruction in brown bears. \u003cem\u003eEcol. Evol.\u003c/em\u003e 12, e9246 (2022).\u003c/li\u003e\n\u003cli\u003eShiretoko Nature Foundation. https://www.shiretoko.or.jp/report/2023/09/7579.html (accessed 3 December 2025).\u003c/li\u003e\n\u003cli\u003eShimizu, K. et al. Seasonal infestation patterns of ticks on Hokkaido sika deer (\u003cem\u003eCervus nippon yesoensis\u003c/em\u003e). \u003cem\u003eParasitology\u003c/em\u003e 151, 1317\u0026ndash;1325 (2024).\u003c/li\u003e\n\u003cli\u003eIto, M. et al. Environmental and host factors underlying tick infestation in invasive raccoons (\u003cem\u003eProcyon lotor\u003c/em\u003e) in Hokkaido, Japan. \u003cem\u003eTicks Tick Borne Dis.\u003c/em\u003e 15, 102389 (2024).\u003c/li\u003e\n\u003cli\u003eKovba, A. et al. No evidence of SARS-CoV-2 infection in urban wildlife of Hokkaido, Japan. \u003cem\u003eTransbound. Emerg. Dis.\u003c/em\u003e 2024, 1204825 (2024).\u003c/li\u003e\n\u003cli\u003eShimozuru, M. et al. Reproductive parameters and cub survival of brown bears in the Rusha area of the Shiretoko Peninsula, Hokkaido, Japan. \u003cem\u003ePLoS One\u003c/em\u003e 12, e0176251 (2017).\u003c/li\u003e\n\u003cli\u003eYamaguti, N. et al. Ticks of Japan, Korea, and the Ryukyu Islands. \u003cem\u003eBrigham Young Univ. Sci. Bull. Biol. Ser.\u003c/em\u003e 15, 1 (1971).\u003c/li\u003e\n\u003cli\u003eTorii, S. et al. Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles. \u003cem\u003eTicks Tick Borne Dis\u003c/em\u003e. 10, 328-335 (2019).\u003c/li\u003e\n\u003cli\u003eMinh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e 37, 2461 (2020).\u003c/li\u003e\n\u003cli\u003eKalyaanamoorthy, S. et al. ModelFinder: fast model selection for accurate phylogenetic estimates. \u003cem\u003eNat. Methods\u003c/em\u003e 14, 587\u0026ndash;589 (2017).\u003c/li\u003e\n\u003cli\u003eGuindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. \u003cem\u003eSyst. Biol.\u003c/em\u003e 59, 307\u0026ndash;321 (2010).\u003c/li\u003e\n\u003cli\u003eHoang, D. T. et al. UFBoot2: improving the ultrafast bootstrap approximation. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e 35, 518\u0026ndash;522 (2018).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Phenuivirus RT-PCR screening in Hokkaido wildlife.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"532\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTissue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRT-PCR (positive/tested)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 115px;\"\u003e\n \u003cp\u003eRaccoon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eLymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e0/94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eSpleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e0/100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 115px;\"\u003e\n \u003cp\u003eSika deer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eLymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e0/7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eSpleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e0/28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003eBrown bear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eSpleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e9/149\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 2. Antibody screening for TOYV in wild animals inhabiting Hokkaido, Japan (2022-2025).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"659\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrder\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies (ENG)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies (Latin)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTested\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeroprevalence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtotal IFA+/tested (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal IFA+/tested (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eELISA +\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIFA +\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody prevalence % by IFA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"11\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarnivora\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" style=\"width: 71px;\"\u003e\n \u003cp\u003eHokkaido brown bear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eUrsus arctos lasiotus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 62px;\"\u003e\n \u003cp\u003eShari\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 69px;\"\u003e\n \u003cp\u003e13*/131 (9.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"6\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u0026nbsp;19/208 (9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 62px;\"\u003e\n \u003cp\u003eRausu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 69px;\"\u003e\n \u003cp\u003e6**/77 (7.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" style=\"width: 71px;\"\u003e\n \u003cp\u003eRaccoon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eProcyon lotor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 62px;\"\u003e\n \u003cp\u003eSapporo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e25.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 69px;\"\u003e\n \u003cp\u003e58/179 (32.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u0026nbsp;58/210 (27.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e39.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e29.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAsahikawa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0/31 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtiodactyla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" style=\"width: 71px;\"\u003e\n \u003cp\u003eHokkaido sika deer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eCervus nippon yesoensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 62px;\"\u003e\n \u003cp\u003eSapporo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 69px;\"\u003e\n \u003cp\u003e2/44 (4.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" style=\"width: 69px;\"\u003e\n \u003cp\u003e2/161 (1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAsahikawa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0/82 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eShiretoko National Park\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0/35 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cem\u003eRodentia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eLarge Japanese field mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eApodemus speciosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCentral Hokkaido\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0/60 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0/187 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eJapanese red-backed vole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eCraseomys rufocanus bedfordiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCentral Hokkaido\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0/52 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eSmall Japanese field mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eApodemus argenteus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCentral Hokkaido\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0/69 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eJapanese Northern red-backed Vole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eClethrionomys rutilus mikado\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCentral Hokkaido\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0/6 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Including 5 RT-PCR-positive bears.\u003c/p\u003e\n\u003cp\u003e**Including 1 RT-PCR-positive bear.\u003c/p\u003e\n\u003c/br\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 is available in the Supplementary Files section.\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"virus, brown bear, phenuivirus, tick-borne, Japan, Hokkaido, wildlife","lastPublishedDoi":"10.21203/rs.3.rs-9045918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9045918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tick-borne phenuiviruses are a group of zoonotic pathogens causing severe, sometimes lethal, diseases in humans. While several novel phenuiviruses have been identified in ticks, especially in East Asia, their mammalian infectivity largely remains uncharacterized. Here, we report the identification and successful isolation of an emerging tick-borne phenuivirus, Toyo virus (TOYV), from Hokkaido brown bears (Ursus arctos yesoensis) during wildlife surveillance in northern Japan. TOYV was isolated from bear samples using type I and II interferon receptor-knockout (AG129) mice and Hep3B cells. Intracerebral inoculation of suckling AG129 mice induced neurological signs, and viral replication was confirmed in mouse neuroblastoma cells. Detection of TOYV RNA in brown bears and Haemaphysalis species ticks, together with seropositivity in brown bears, raccoons, and sika deer, demonstrated active tick-borne circulation of TOYV among diverse wildlife hosts. This study provides the first evidence of tick-borne phenuivirus infections in an ursid species and the neuroinvasive and neurotropic potential of TOYV. Our findings underscore the critical role of wildlife surveillance in early detection of viruses with zoonotic potential and accelerating responses to emerging viral epidemics.","manuscriptTitle":"Circulation of an emerging neurotropic tick-borne phenuivirus in brown bears and wildlife in northern Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 04:34:37","doi":"10.21203/rs.3.rs-9045918/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"73fb05aa-d2ab-406e-8b38-a176b666feaf","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64651350,"name":"Biological sciences/Microbiology/Virology/Viral epidemiology"},{"id":64651351,"name":"Biological sciences/Microbiology/Pathogens"},{"id":64651352,"name":"Biological sciences/Microbiology/Virology/Viral reservoirs"}],"tags":[],"updatedAt":"2026-03-18T04:34:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 04:34:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9045918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9045918","identity":"rs-9045918","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}