{"paper_id":"200f891d-c97c-4760-839b-24f96ec8ec7d","body_text":"Historic and contemporary museum specimens implicate Northern Red-backed Vole 1 \n(Clethrionomys rutilus) as borealpox host as early as 1990s 2 \n 3 \nMaya Juman*1,2, Jeffrey B. Doty*3,6, Clint N. Morgan3, Audrey Matheny3, Ariel Caudle3, 4 \nMarissa Breslin2, Natalie M. Hamilton2, Aren Gunderson2, Katherine Newell4,5, Julia Rogers4,5, 5 \nVictoria A. Balta5,6, Italo B. Zecca3,5, Florence Whitehill3, Faisal S. Minhaj3, Molly M. 6 \nMcDonough7,8, Adam Ferguson8, Yu Li3, Crystal Gigante3, Yoshinori Nakazawa3, Joseph 7 \nMcLaughlin4, Link E. Olson2 8 \n  9 \n1Department of Veterinary Medicine, University of Cambridge, UK 10 \n2Department of Mammalogy, University of Alaska Museum, University of Alaska Fairbanks, 11 \nFairbanks, AK, USA 12 \n3Poxvirus and Rabies Branch, Division of High-Consequence Pathogens and Pathology, National 13 \nCenter for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and 14 \nPrevention, Atlanta, GA, USA 15 \n4Alaska Division of Public Health, Section of Epidemiology, Anchorage, AK, USA 16 \n5Epidemic Intelligence Service, Centers for Disease Control and Prevention, Atlanta, Georgia, 17 \nUSA. 18 \n6Arctic Investigations Program, Division of Infectious Disease Readiness and Innovation, 19 \nNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and 20 \nPrevention, Anchorage, AK, USA 21 \n7Department of Biological Sciences, Chicago State University, Chicago, IL, USA 22 \n8Gantz Family Collections Center, Field Museum of Natural History, Chicago, IL, USA 23 \n  24 \n*These authors contributed equally to this work. 25 \n 26 \nAbstract 27 \n 28 \nBorealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported 29 \nhuman infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of 30 \nBRPV is unknown, although previous investigations have raised the possibility of wild small 31 \nmammals transmitting the virus to humans, either through direct contact or via domestic cats and 32 \ndogs. To understand which species may be involved in the maintenance and/or spillover of 33 \nBRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and 34 \nsquirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai 35 \nPeninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five 36 \nspecies (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable 37 \nvirus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from 38 \nhistorical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali 39 \nNational Park and Preserve in 1998 and 1999, 17 years before the first reported human case of 40 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nBRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the 41 \nhypothesis of local human infections through multiple spillover events. These findings suggest 42 \nC. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in 43 \nAlaskan wild small-mammal populations for at least 25 years. Our study highlights the potential 44 \nof museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. 45 \nFurther museum- and field-based sampling will clarify the true geographic range of BRPV, 46 \nwhich is closely related to Old World orthopoxviruses and may be circulating beyond North 47 \nAmerica. 48 \n 49 \nIntroduction 50 \n  51 \nThe genus Orthopoxvirus (family Poxviridae) contains ten recognized species of double-stranded 52 \nDNA viruses that cause disease in a wide range of mammalian hosts, including humans (Bonwitt 53 \net al. 2022). Notably, orthopoxviruses (OPXVs) include variola virus (VARV), which causes 54 \nsmallpox, and monkeypox virus (MPXV), the causative agent of monkeypox, which has recently 55 \ncaused widespread human outbreaks with sustained human-to-human transmission (Bonwitt et 56 \nal. 2022; WHO 2022). OPXVs may be emerging at higher rates due to increased host mixing and 57 \nhuman-wildlife contact driven by climate and land-use change (Thomassen et al. 2013), as well 58 \nas heightened human susceptibility following the cessation of smallpox vaccination after its 59 \nglobal eradication in 1980 (Diaz 2021). 60 \n 61 \nThe most recently described OPXV is borealpox virus (BRPV), formerly known as Alaskapox 62 \nvirus (Gigante et al. 2019). BRPV was first detected after a patient presented with a lesion near 63 \nFairbanks, Alaska, USA in July 2015 (Springer et al. 2017). Over the following eight years, five 64 \nadditional, epidemiologically unrelated cases were reported in Fairbanks, all involving self-65 \nlimiting illness (Mooring et al. 2020; Mooring et al. 2021; Mooring et al. 2025). However, in 66 \nSeptember 2023, the first fatal case was reported in an elderly, immunocompromised patient on 67 \nthe Kenai Peninsula in Southcentral Alaska (~320 miles straight-line distance SSE of Fairbanks), 68 \nexpanding the known geographic range of this virus and demonstrating its potential for causing 69 \nfatal outcomes, particularly in severely immunocompromised individuals (Rogers et al. 2025). 70 \nFurthermore, the viral sequence recovered from this patient was distinct from sequences isolated 71 \nfrom prior cases in the Fairbanks area (Rogers et al. 2025), suggesting genetic variation across 72 \nspace as is the case in OPXVs with wide host ranges such as cowpox virus (CPXV) and zoonotic 73 \nMPXV (Reynolds et al. 2018). Unraveling the geographic origins and range, host species, and 74 \ntransmission pathways of BRPV is important for understanding and mitigating this viral 75 \npathogen. 76 \n 77 \nIn the initial 2015 investigation of the first BRPV case, inadvertent viral importation from 78 \noutside of Alaska could not be excluded (Springer et al. 2017). However, the subsequent cases in 79 \nthe Fairbanks area, the patients’ lack of out-of-state travel, and the greater genetic diversity seen 80 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nin the Kenai isolate strongly indicate that the virus is circulating in at least one non-human 81 \nreservoir in Alaska. This is particularly noteworthy given the phylogenetic relationship between 82 \nBRPV and other OPXVs. The genus Orthopoxvirus contains two reciprocally monophyletic 83 \nclades: “Old World” and “New World” OPXVs, which are thought to have diverged from a 84 \ncommon ancestor approximately 42,000 years ago (Babkin et al. 2022). Despite being detected 85 \nonly in Alaska, BRPV isolates share a most recent common ancestor with Old World OPXVs 86 \nand constitute a reciprocally monophyletic sister clade with an estimated divergence date of 87 \napproximately 19,000 years ago (Babkin et al. 2022). The geographic range of BRPV remains 88 \nunknown, and it may be circulating beyond Alaska and North America. 89 \n  90 \nThe natural reservoir (sensu Viana et al. 2014) of BRPV is also unknown, but evidence from 91 \ncases, wildlife screening, and the genomic diversity within BRPV suggests small mammals play 92 \na role (Mooring et al. 2025). All known patients lived in low population-density, forested areas 93 \nand reported contact with domestic animals (i.e., dogs and cats), many of which were known to 94 \nhunt small mammals such as rodents and shrews (i.e., pet dogs and cats that purportedly hunted 95 \nsmall mammals) (Springer et al. 2017; Mooring et al. 2020; Mooring et al. 2021; Mooring et al. 96 \n2025; Rogers et al. 2025). Furthermore, all reported cases occurred in the late summer to early 97 \nfall, when outdoor recreational and subsistence activities (e.g., hunting and foraging) increase 98 \nacross Alaska; three out of the seven patients reported berry picking prior to symptom onset. 99 \nThese activities increase opportunities for direct wildlife contact or indirect exposure via fomites, 100 \nboth established pathways for OPXV transmission (Bonwitt et al. 2022). In 2015, samples from 101 \nsmall mammals collected near the first patient’s home were screened for OPXV DNA by PCR 102 \n(serological testing was not conducted); all samples were negative (Springer et al. 2017). During 103 \na 2020 investigation following a reported case in the Fairbanks area, 176 small mammals were 104 \ncaptured and sampled. Anti-OPXV IgG antibodies were detected in serum samples from 28 of 105 \n147 Northern Red-backed Voles (Clethrionomys rutilus; 19.0%), one of four Northern Flying 106 \nSquirrels (Glaucomys sabrinus; 25.0%), and one of three American Red Squirrels (Tamiasciurus 107 \nhudsonicus; 33.3%) (Mooring et al. 2025). Viral DNA was amplified from tissue samples of 12 108 \nof 149 Northern Red-backed Voles (8.1%) and one of 14 Masked Shrews (Sorex cinereus; 109 \n7.1%). Among PCR-positive samples, viable BRPV was detected in one Northern Red-backed 110 \nVole and one Masked Shrew. 111 \n 112 \nThe emergence of BRPV in North America raises pressing questions about its origins and actual 113 \ngeographic distribution, which additional field-based sampling can help address. A 114 \ncomplementary approach involves the screening of historical specimens in natural history 115 \nmuseums. These collections are valuable but largely underutilized resources for pathogen 116 \nreservoir identification, with the potential to expand the known spatial, taxonomic, and temporal 117 \ndistributions of viruses of concern (Yates et al. 2002; Colella et al. 2021). In this study, we aimed 118 \nto identify the reservoir host(s) for BRPV through both field- and museum-based screening. 119 \nFirst, we sampled small mammals in and around the properties of human cases and other non-120 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nepidemiologically linked locations near Fairbanks and Kenai, Alaska. Here, we sought to expand 121 \nthe taxonomic and geographic scope of previous sampling efforts (Springer et al. 2017; Mooring 122 \net al. 2025) by screening a larger sample of small mammals, following similar field studies of 123 \nMPXV and Akhmeta virus, another recently described OPXV (Doty et al. 2017; Doty et al. 124 \n2019). To broaden the temporal scope, we also screened tissue samples from historical 125 \nspecimens housed at the University of Alaska Museum (UAM), targeting species based on 126 \npreliminary results from field-based sampling (Mooring et al. 2025). 127 \n 128 \nResults 129 \n 130 \nSmall mammals trapped in 2021 and 2024 131 \n 132 \nIn September 2021, 202 wild small mammals representing at least six species were trapped and 133 \nsampled in the Fairbanks and North Pole regions of Interior Alaska (Table 1). The majority were 134 \nNorthern Red-backed Voles (n = 147), followed by shrews (Sorex spp.; 21), American Red 135 \nSquirrels (15), Northern Flying Squirrels (12), voles (Microtus sp.; 6), and a single Woodchuck 136 \n(Marmota monax). Additionally, a series of domestic/commensal rats (Rattus sp.; 7) obtained by 137 \nthe Alaska Department of Fish and Game (and subsequently deposited in the UAM’s Mammal 138 \nCollection) from the Fairbanks area were also sampled. In August 2024, 135 wild small 139 \nmammals of at least six species were trapped and sampled in the Kenai Peninsula area of Alaska 140 \n(Table 1). Similarly, the majority were Northern Red-backed Voles (83), followed by shrews 141 \n(37), American Red Squirrels (11), Northern Bog Lemmings (Synaptomys borealis; 2), one 142 \nHouse Mouse (Mus musculus), and one Ermine (Mustela erminea). 143 \n 144 \nIn 2021, 20 Northern Red-backed Voles (13.6%) were IgG-positive for OPXV antibodies by 145 \nELISA, as were five American Red Squirrels (33.3%) and seven Northern Flying Squirrels 146 \n(58.3%). However, OPXV DNA was only detected in seven Northern Red-backed Voles (4.8%). 147 \nIn 2024, seven Northern Red-backed Voles (8.4%) were IgG-positive for OPXV antibodies by 148 \nELISA, as were one shrew (2.7%), one American Red Squirrel (9.1%), and one House Mouse 149 \n(100%). Once again, the only PCR-positive animal was a Northern Red-backed Vole (1.2% of 83 150 \nC. rutilus). Viable virus was isolated from one PCR-positive Northern Red-backed Vole sample 151 \ncollected in 2024. Sequencing was conducted on this viral isolate and one PCR-positive Northern 152 \nRed-backed Vole sample from 2021. None of the PCR-positive animals from either sampling 153 \nyear had visible lesions suggestive of a poxvirus infection. 154 \n 155 \n 156 \n 157 \n 158 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nTable 1. Results of serological assays, PCR assays, and viral culture attempts on samples taken 159 \nfrom wild small mammals trapped in 2021 and 2024. A breakdown of results by trap site is 160 \nincluded in Supplementary Data S1. 161 \n 162 \n 163 \nHistorical voucher specimen investigations 164 \n 165 \nWe screened a total of 285 frozen tissue samples from vole, shrew, and squirrel specimens 166 \nhoused at UAM for OPXV DNA. Four of the 201 samples of Northern Red-backed Voles were 167 \npositive for OPXV DNA (Table 2). Two of these four PCR-positive samples were taken from the 168 \nsame vole specimen (UAM 51567), and both yielded viable virus (Table 3). All three specimens 169 \n(UAM 51528, UAM 51567, UAM 73062) were collected at Rock Creek Site in Denali National 170 \nCommon name Species Sample \nsize \nELISA \npositives (%) \nPCR positives \n(%) \nViable \nvirus (%\nFairbanks and North Pole, Alaska; September 2021 \nNorthern Red-backed Vole Clethrionomys rutilus 147 20 (13.6) 7 (4.8) 0 \nShrew Sorex spp. 21 0 0 0 \nAmerican Red Squirrel Tamiasciurus hudsonicus 15 5 (33.3) 0 0 \nNorthern Flying Squirrel Glaucomys sabrinus 12 7 (58.3) 0 0 \nRat Rattus sp. 7 0 0 0 \nVole Microtus sp. 6 0 0 0 \nWoodchuck Marmota monax 1 0 0 0 \nTotals 209 32 (15.3) 7 (3.3) 0 \nKenai Peninsula, Alaska; August 2024 \nNorthern Red-backed Vole Clethrionomys rutilus 83 7 (8.4) 1 (1.2) 1 (1.2)  \nShrew Sorex spp. 37 1 (2.7) 0 0 \nAmerican Red Squirrel Tamiasciurus hudsonicus 11 1 (9.1) 0 0 \nNorthern Bog Lemming Synaptomys borealis 2 0 0 0 \nHouse Mouse Mus musculus 1 1 (100.0) 0 0 \nErmine Mustela erminea 1 0 0 0 \nTotals 135 10 (7.4) 1 (0.7) 1 (0.7)  \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nPark and Preserve, Alaska, in July 1998, August 1998, and August 1999, respectively (Fig. 1). A 171 \nfrozen liver sample was positive from each specimen, as well as a frozen mixed-tissue sample 172 \nfrom UAM 51567. Frozen spleen and mixed-tissue samples from UAM 73062 were also 173 \nscreened, and neither was positive for OPXV DNA. 174 \n 175 \nTable 2. Results of PCR and viral culture attempts on samples from museum voucher specimens. 176 \nCollection date, locality, and tissue type of each sample are included in Supplementary Data S1. 177 \n 178 \nCommon name Species Samples  PCR positives (%) Viable virus \n(%) \nNorthern Red-backed Vole Clethrionomys rutilus 201 4 (2.0) 2 (1.0) \nTundra Vole Alexandromys \noeconomus \n22 0 0 \nInsular Vole Microtus abbreviatus 6 0 0 \nVole Microtus sp. 1 0 0 \nMasked Shrew Sorex cinereus 43 0 0 \nTundra Shrew Sorex tundrensis 8 0 0 \nMontane Shrew Sorex monticolus 3 0 0 \nAmerican Red Squirrel Tamiasciurus hudsonicus 1 0 0 \nTotals 285 4 (1.4) 2 (0.7) \n 179 \n  180 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nTable 3. Tissue type, collection date, and locality of OPXV PCR-positive Northern Red-backed 181 \nVole (Clethrionomys rutilus) museum voucher specimens. 182 \n 183 \n 184 \n 185 \nFig. 1. Localities of museum specimens sampled in our study. Diamonds indicate PCR-positive 186 \nspecimens and smaller black dots indicate all other sampling sites. 187 \n 188 \n 189 \nCatalog \nnumber \nTissue type Viable virus \nisolated? \nLocality Coordinates  Collection \ndate \nSex \nUAM 51528 Liver No \nRock Creek Site, \nDenali National \nPark, Alaska \n63.729636, -\n148.982167 \n21 July \n1998 Female \nUAM 51567 \nLiver Yes Rock Creek Site, \nDenali National \nPark, Alaska \n63.727261, -\n148.980936 \n20 August \n1998 Male \nMixed tissue Yes \nUAM 73062 Liver No \nRock Creek Site, \nDenali National \nPark, Alaska \n63.730683, -\n148.984308 \n31 August \n1999 Male \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nPhylogenetic analysis of borealpox virus whole-genome sequences 190 \n 191 \nA whole-genome maximum-likelihood tree revealed that isolates from Fairbanks human cases 192 \n(2015-2023) form a well-supported clade with small mammal specimens from Fairbanks C. 193 \nrutilus (2020, 2021) and S. cinereus (2020) sampled in this and an earlier study (Mooring et al. 194 \n2025). There was also strong support for a separate clade containing isolates from the 2023 195 \nKenai human case and C. rutilus sampled from Kenai in 2024. The BRPV isolate from the C. 196 \nrutilus specimen collected in Denali National Park and Preserve in 1998 (UAM 51567) fell 197 \nwithin the Fairbanks clade, clustering with human cases from 2022 and 2023 (Fig. 2). Notably, 198 \nboth Bayesian and maximum-likelihood analyses did not support monophyly of human-derived 199 \nsequences, suggesting multiple separate spillover events.  200 \n 201 \n 202 \n203 \nFig. 2. Maximum-likelihood tree based on whole-genome sequences from BRPV isolates, 204 \nconstructed using a GTR+F model in IQ-TREE (Nguyen et al. 2015). Blue labels indicate human 205 \nand vole isolates from the Kenai Peninsula, while the red label indicates the isolate from UAM 206 \n51567. The vole sequence from Fairbanks 2021 was sequenced directly from DNA extracted 207 \nfrom tissue. Bootstrap support values are shown at each node. 208 \n 209 \nDiscussion 210 \n \nan \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \n 211 \nBorealpox virus (BRPV) is a recently identified OPXV that has repeatedly spilled over into 212 \nhuman populations in Alaska since first reported in 2015. To better understand the zoonotic 213 \nreservoir of BRPV, we trapped and sampled wild small mammals in areas of known BRPV 214 \nspillover in Alaska—the Interior Region’s Fairbanks region in September 2021 and the Kenai 215 \nPeninsula in 2024—and screened tissue and blood samples for OPXV DNA and OPXV 216 \nantibodies, respectively. We also screened tissue samples associated with historical museum 217 \nspecimens of suspected host species (collected 1991-2019 and housed at UAM) for OPXV DNA. 218 \n 219 \nAs in earlier field-based surveys (Mooring et al. 2025), most seropositive animals in our study 220 \nwere Northern Red-backed Voles (n = 27), with 13.6% seropositivity and 8.4% seropositivity for 221 \nOPXV antibodies in Fairbanks (2021) and Kenai (2024), respectively. American Red Squirrels, 222 \nNorthern Flying Squirrels, an unspecified shrew, and a House Mouse were also seropositive 223 \n(Table 1). The IgG ELISA used in this study is not BRPV-specific, so these results may reflect 224 \npast infection with other OPXVs, which could potentially circulate undetected in these small-225 \nmammal populations; however, no other OPXVs have been identified to date in Alaska. Other 226 \nOPXVs thought to circulate in North American small mammals have not been detected in 227 \nAlaska, including raccoonpox, volepox, and skunkpox viruses (Alexander et al. 1972; Regnery 228 \n1987; Emerson et al. 2009). Consequently, the detection of OPXV antibodies in a House Mouse 229 \n(M. musculus) is notable; should this reflect a prior BRPV infection, it suggests the potential for 230 \nmouse-human transmission risk in domestic settings, which may be higher than the risk of 231 \ntransmission from other small mammals that are less likely to be found in human dwellings. The 232 \ndistribution and prevalence of M. musculus in Alaska is very poorly understood, but museum 233 \nvoucher specimens have been collected from or near Fairbanks, Fort Yukon, Anchorage, the 234 \nKenai Peninsula, Kodiak Island, St. Paul Islands, Unalaska Island, Kiska Island, and multiple 235 \nlocalities in Southeast Alaska (MacDonald and Cook 2009). Predictive modeling based on viral 236 \ngenomic features has implicated the genus Mus as potentially suitable borealpox hosts (Tseng et 237 \nal. 2025). Expanded serological and molecular testing of mice in and around the homes of BRPV 238 \npatients, and throughout Alaska, will be critical for investigating this hypothesis. 239 \n 240 \nOnly Northern Red-backed Voles tested positive for OPXV DNA (seven in 2021 and one in 241 \n2024), two of which were confirmed as BRPV by DNA sequencing (Figure 2.) Viable virus was 242 \nisolated from the positive sample collected in 2024. This supports prior evidence that the 243 \nNorthern Red-backed Vole, which is also commonly found in and around human dwellings, is 244 \ninvolved in the maintenance and circulation of BRPV in wildlife populations in Alaska (Mooring 245 \net al. 2025). To further investigate the reservoir of BRPV, future research should include 246 \nlongitudinal study of potential host species, confirmation of long-term viral maintenance in these 247 \npopulations, and expanded screening of species beyond Northern Red-backed Voles. Our study 248 \nis limited by small sample sizes of most species and a heavy bias toward C. rutilus based on the 249 \navailability of samples to screen and previous evidence of BRPV circulation in this species. 250 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nOther species—including squirrels, a mouse, and a shrew—were seropositive in our study, and 251 \nthe screening of larger sample sizes of these species may reveal evidence of PCR positivity and 252 \nviable virus. A recent study suggests squirrels as possible reservoirs of MPXV, a related OPXV 253 \n(Riutord-Fe et al. 2026), and future work on BRPV should continue to investigate the possibility 254 \nof other small mammal species playing a role in its maintenance and transmission. 255 \n 256 \nWhile future field studies of BRPV dynamics in wild mammals will be critical for elucidating 257 \nthe reservoir species, looking for evidence of past BRPV circulation in Alaska can complement 258 \ncontemporary data and further our understanding of the ecology of this virus. To do this, we 259 \nscreened 285 tissue samples from historical museum specimens of voles, shrews, and squirrels 260 \nfor OPXV DNA, and found four positive samples—all from Northern Red-backed Voles (Table 261 \n2). DNA sequencing confirmed the presence of BRPV DNA in one of these samples (Figure 2.). 262 \nPositive samples were collected from Denali National Park and Preserve in 1998 and 1999, over 263 \n15 years before the first reported case of human BRPV infection (Table 3). Denali National Park 264 \nand Preserve is roughly equidistant from the known spillover locations of BRPV (Fairbanks and 265 \nKenai Peninsula) (Fig. 1). The detection of BRPV DNA in samples from the 1990s and in a 266 \ndifferent site from known cases confirms the wider circulation of BRPV in Alaskan small-267 \nmammal populations over a longer period of time than previously known. Phylogenetically, the 268 \nisolate from this museum specimen fell within a clade of contemporary human and small-269 \nmammal isolates from the Fairbanks area (Fig. 2). Another separate, reciprocally monophyletic 270 \nclade contains isolates from the Kenai Peninsula human case in 2023 and C. rutilus sampled 271 \nfrom case patient’s residence in 2024, suggesting that the Kenai patient was likely infected 272 \nlocally (Fig. 2). Our findings support the hypothesis of a long-term, established wildlife reservoir 273 \nwith occasional spillover into human populations and potentially other mammal populations. 274 \nInitial investigations of human cases could not exclude the possibility of recent BRPV 275 \nintroduction into Alaska (Springer et al. 2017). However, our study suggests that BRPV has been 276 \npresent in Alaska for at least 25 years (and likely much longer) with multiple separate spillover 277 \nevents since 2015, though the drivers of these events remain unknown. It is possible that 278 \nprevious spillover events went undetected due to mild, self-limiting illness or symptoms being 279 \nmistaken for other rashes (Mooring et al. 2020; Mooring et al. 2021). Alternatively, spillover 280 \nmay have become more likely in the past decade due to the effects of climate and land-use 281 \nchange on the distributions and cross-species interactions of small mammals (Baltensperger et al. 282 \n2024), as is the case for MPXV (Thomassen et al. 2013), and/or waning population-level 283 \nimmunity against OPXVs following the cessation of smallpox vaccination (Diaz 2021). 284 \n 285 \nThe Northern Red-backed Vole is found in Alaska, Canada, northern Russia, and Scandinavia 286 \n(Linzey et al. 2020). If this species is the primary reservoir of BRPV, our findings suggest that 287 \nBRPV may be circulating beyond North America, in C. rutilus as well as in related species that 288 \nhybridize with C. rutilus in contact zones (Runck et al. 2009; Wiens and Colella 2025). A 289 \nbroader awareness of BRPV symptoms and diagnostics will be critical to identify other spillover 290 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nevents in Alaska and the broader arctic region. Further molecular and serological surveillance of 291 \nwild mammal populations in Alaska, as well as longitudinal studies of suspected host species 292 \nlike Northern Red-backed Voles, will be vital to identifying the drivers of the apparently sudden 293 \nspillover of BRPV into human populations.  294 \n 295 \nWhen federal public health agencies partner with non-governmental organizations such as 296 \nnatural history museums and academic institutions, investigations are strengthened by leveraging 297 \nthe resources, capabilities, and expertise of all partners. Our study illustrates the value of 298 \nmuseum specimens and their unique potential to elucidate the origins, reservoirs, and dynamics 299 \nof emerging viruses. As demonstrated here, disease surveillance in historical museum collections 300 \ncan expand our understanding of the temporal, spatial, or host taxonomic range of newly 301 \ndiscovered pathogens (Juman et al. 2025; Cronin et al. 2025). This work can clarify whether a 302 \nvirus was recently introduced to a given region or whether it has been endemic for decades and 303 \npresent in additional nearby areas, as is the case for BRPV in Alaska. The continued collection 304 \nand preservation of voucher specimens will allow future researchers to retrospectively 305 \nunderstand pathogen emergence in the context of unprecedented global change. 306 \n 307 \nMethods 308 \nSmall-mammal trapping and specimen collection 309 \nSmall mammals were trapped at nine sites around the Fairbanks and North Pole regions of 310 \nAlaska during September 6-14, 2021 (Fig. 1). Sites included homes of the two human cases 311 \nreported in 2021 and locations within seven miles of the case’s home where permission was 312 \nreceived from landowners. Other potential exposure locations such as homes of family members 313 \nwere also included. All sites were in mixed evergreen and deciduous forests, either on public 314 \nland (n = 2) or low-density residential areas (n = 7). 315 \nFollowing the identification of the BRPV case on the Kenai Peninsula in late 2023, small 316 \nmammals were also trapped at three sites in the Kenai/Soldotna/Sterling area during August 15-317 \n22, 2024 (Fig. 1). One site was on public land north of the Kenai Municipal Airport and was 318 \npartially boggy while the other two sites were in low-density residential areas with mixed 319 \nevergreen and deciduous forests, including the home of the case patient. 320 \nTrapping was primarily conducted using standard (7.6 x 8.9 x 22.9 cm) Sherman live traps (H.B. 321 \nSherman Traps, Inc., Tallahassee, FL, USA), which were deployed for a total of 1384 trap-nights 322 \n(i.e., the sum of the number of traps placed each night over the course of trapping) in 2021 and 323 \n1825 trap-nights in 2024. Tomahawk live traps (Model 102, Tomahawk Live Trap, Hazelhurst, 324 \nWI, USA) were used for larger mammals such as squirrels, and pitfall traps were deployed to 325 \ncatch shrews. The Tomahawk and Sherman traps were baited with a mixture of peanut butter and 326 \noats, with carrots included in Fairbanks due to colder temperatures. Trapped animals were 327 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nanesthetized with isoflurane then humanely euthanized and examined prior to examination for 328 \nskin lesions and collection of blood and tissue samples (liver, skin, pooled heart and lung, and 329 \npooled kidney and spleen; lesion material if observed). All trapping and animal handling 330 \nfollowed protocols approved by the CDC and UAF’s Institutional Animal Care and Use 331 \nCommittees (CDC: 3183DOTMULX, 3400DOTMULX; UAF: 152295). Tissue specimens were 332 \nalso obtained from roadkill animals collected by the UAM researchers and from animals 333 \ncollected by the Alaska Department of Fish and Game. All voucher specimens and their 334 \nassociated tissues have been deposited and catalogued in UAM’s Mammal Collection 335 \n(Supplementary Data S1). 336 \nMuseum specimen sampling 337 \n 338 \nWe screened tissue samples from specimens of voles, shrews, and squirrels archived at UAM. 339 \nThe following species were selected for screening based on the availability of tissue samples, as 340 \nwell as preliminary results about seropositivity and molecular BRPV detection in wild small 341 \nmammals: Northern Red-backed Voles (n = 201 samples), Tundra Voles (Alexandromys 342 \noeconomus; 22), Singing Voles (Microtus miurus; 6), a vole of undetermined species (Microtus 343 \nsp.; 1), Masked Shrews (43), Tundra Shrews (Sorex tundrensis; 8), Montane Shrew (Sorex 344 \nmonticolus; 3), and an American Red Squirrel (1). The specimens were collected at various 345 \nlocations around Fairbanks, Kenai, and Denali National Park and Preserve from 1991-2019 (Fig. 346 \n1; Supplementary Data S1). 347 \n 348 \nHistorical samples archived at UAM were flash-frozen without a buffer and housed in liquid 349 \nnitrogen-cooled cryovats that maintain vapor-phase nitrogen at -170°C. The work surface and 350 \ntools were cleaned with 10% bleach. The box containing the appropriate tissues was removed 351 \nfrom the cryovat freezer and placed on an insulated cold-block while outside the cryovat. Using a 352 \nseparate set of cleaned instruments for each tube, a small subsample (roughly 0.1 gram) of tissue 353 \nwas removed from each cryotube and transferred into appropriately labeled tubes. Between 354 \nsampling, instruments were cleaned in 10% bleach solution and then rinsed with water and dried. 355 \nAfter subsampling, the original samples were returned to the cryovat freezer, and the subsamples 356 \nwere placed in a labeled box on a second insulated cold-block. Once all subsampling was 357 \ncompleted, the box of subsamples was stored in an ultra-low freezer until shipment. Samples 358 \nwere shipped on dry ice. 359 \n 360 \nLaboratory Diagnostics 361 \nTissue samples were homogenized in 500μ l of PBS with 250μ l of 1mm zirconia silica beads 362 \n(Biospec) and a Mini BeadBeater 24 following the manufacturer’s recommendations. 100μ l of 363 \ntissue homogenate was used for DNA extraction with a MagMAX deep-well magnetic processor 364 \n(ThermoFisher Scientific, https://www.thermofisher.com) using the MagMAX DNA Multi-365 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nSample Ultra kit. DNA samples were assessed for the presence of OPXV with the CDC OPXV 366 \ngeneric real-time PCR assay (Li et al. 2006). 367 \nA modified anti-OPXV IgG enzyme-linked immunosorbent assay (ELISA) was conducted on 368 \nsera obtained from cardiac punctures or on dried blood spots collected on Nobuto filter paper 369 \n(Advantec, San Diego, CA) as previously described (Hutson et al. 2009, Doty et al. 2017). 370 \nSamples were considered positive if the OD value passed the cut-off value in at least two 371 \nconsecutive dilutions (1:100 and 1:200). 372 \nViral isolation and DNA sequencing 373 \nFollowing PCR, samples with OPXV DNA amplification were added to cell culture for virus 374 \nisolation. Remaining tissue homogenate from the DNA extraction process was added to BSC-40 375 \ncell monolayers (African Green Monkey Chlorocebus sabaeus kidney cell line) in T-25 cell 376 \nculture flasks and incubated at 37°C with 5% CO2 (Hutson et al. 2013). DNA was then extracted 377 \nfrom propagated virus and sheared to target 500 bp fragments on a Covaris S220 instrument 378 \n(Covaris, Woburn, Massachusetts, USA). The Swift Accel-NGS 2S DNA Library Kit was used 379 \nfor library preparation with dual indexing following the manufacturer’s instructions (Swift 380 \nBiosciences, product no longer available). Libraries were visualized with an Agilent 2200 Tape 381 \nStation (Agilent Technologies, Santa Clara, California, USA), followed by sequencing on an 382 \nIllumina MiSeq machine with the MiSeq Reagent v3 600 cycle kit (Illumina, San Diego, 383 \nCalifornia, USA). 384 \nGenome Assembly 385 \nReads were trimmed to a minimum length of 50 bp and minimum quality 20 with FaQCs v1.34 386 \n(https://github.com/LANL-Bioinformatics/FaQCs), and three nucleotides were removed from 387 \neach end. The 2015 BRPV reference genome (GenBank accession MN240300.1) was used for 388 \nmapping, with bwa mem v0.7.17 (https://github.com/lh3/bwa). Mapped reads were then 389 \nextracted with samtools v.1.9 (https://github.com/samtools/samtools) and de novo assembled 390 \nwith SPAdes v3.13.0 (https://github.com/ablab/spades), using the --careful flag and --cov-cutoff 391 \noff. Contigs with coverage >10 were manually assembled in Geneious Prime 2023.0.4 392 \n(Dotmatics, Boston, Massachusetts, USA) after mapping. Draft genomes were then edited in 393 \nrepeat regions at approximate positions 150, 163, 174, and 200 kb to match repeat lengths from 394 \nthe 2015 BRPV genome, and only the left ITR and 500 bp of the right ITR were retained. 395 \nTrimmed reads were mapped back to the draft genomes, and a polished genome was generated 396 \nwith ivar v0.1, using the conditions above except requiring a minimum read depth of 10. Finally, 397 \nto produce a final genome, ITRs were copied from the left to the right end. Annotations from the 398 \nreference genome were transferred using Liftoff (https://github.com/agshumate/Liftoff).  399 \n 400 \nPhylogenetic Analysis 401 \n 402 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint \n\n \nGenome alignments were performed with MAFFT v7.490 in Geneious Prime 2023.0.4 using 403 \nFFT-NS-i x2 and final alignments are provided as supplementary files (Supplementary Data S2). 404 \nBayesian phylogenetic trees were generated in BEAST v2.7.7 (Bouckaert et al. 2019) in two runs 405 \nwith the following parameters based on Gigante et al. (2019): GTR+I+G nucleotide substitution 406 \nmodel (4 gamma categories, 35% invariant), relaxed lognormal clock (exponential distribution of 407 \nucldStdev prior with mean = 0.333), and Yule model prior until all parameters exhibited ESS > 408 \n200 after 10% burn-in, visualized in Tracer. Default parameters were used unless specified. Run 409 \nlog and tree files were combined using LogCombiner after 10% burn-in. A maximum clade 410 \ncredibility tree was estimated in TreeAnnotator based on sampling frequency of 1000 and 10% 411 \nburn-in. We also estimated a maximum-likelihood tree on the whole genome using IQ-TREE 412 \n(Nguyen et al. 2015), with branch supports obtained using the ultrafast bootstrap method and 413 \n1000 replicates. The nucleotide substitution model for the maximum-likelihood tree (GTR+F) 414 \nwas determined using ModelFinder implemented through IQ-TREE (Kalyaanamoorthy et al. 415 \n2017). Vaccinia virus (GenBank AY603355) was used as an outgroup. 416 \n 417 \nAcknowledgements 418 \n 419 \nThis research was supported by generous donations to the University of Alaska Museum’s 420 \nMammal Collection from the Jay Pritzker Foundation and Nancy Eliason. MMJ was supported 421 \nby a Gates Cambridge Scholarship enabled by grant OPP1144 from the Bill & Melinda Gates 422 \nFoundation. CNM, AM, and AC were supported in part by appointment to the Research 423 \nParticipation Program at the Centers for Disease Control and Prevention, administered by the 424 \nOak Ridge Institute for Science and Education through an interagency agreement between the 425 \nU.S. Department of Energy and CDC. MMM was supported by an NSF CAREER Award 426 \n(2238801). The authors thank Mallory Gulbranson and Kyndall B.P. Hildebrandt at the 427 \nUniversity of Alaska Museum for access to frozen tissues in their care and assistance with 428 \nsubsampling. The authors also thank Nicholas Fowler from Alaska Department of Fish and 429 \nGame for his help with accessing sampling sites on the Kenai Peninsula. 430 \n 431 \nDisclaimer: The findings and conclusions in this report are those of the authors and do not 432 \nnecessarily represent the official position of the Centers for Disease Control and Prevention. 433 \n 434 \nReferences 435 \n 436 \nAlexander, A. D., et al. 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The ecology and evolutionary history of an emergent disease: Hantavirus 552 \npulmonary syndrome. BioScience 52, 989 (2002). 553 \n105 and is also made available for use under a CC0 license. \n(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC \nThe copyright holder for this preprintthis version posted March 25, 2026. ; https://doi.org/10.64898/2026.03.22.713527doi: bioRxiv preprint","source_license":"Public-Domain","license_restricted":false}