Historic and contemporary museum specimens implicate Northern Red-backed Vole (Clethrionomys rutilus) as borealpox host as early as 1990s

This study identifies the Northern Red-backed Vole (*Clethrionomys rutilus*) as a likely reservoir for Borealpox virus by detecting the pathogen in both contemporary wild populations and historical museum specimens dating back to the late 1990s, suggesting the virus has been circulating in Alaskan small mammals for at least 25 years prior to the first reported human cases.

The Gist

The Case of the Missing Virus: How Museum Specimens Solved a 25-Year Mystery

Imagine Alaska as a vast, quiet library. For years, a strange and dangerous "book" (a virus called Borealpox) has been causing trouble for a few people who live there. Doctors knew the virus existed because it made people sick, sometimes even fatally, but they had no idea where it was hiding in nature or how long it had been there. It was like finding a fire in a forest but having no idea where the spark started or how long the embers had been smoldering.

This paper is the story of how scientists became detectives, using two powerful tools: live trapping (catching animals in the present) and museum archives (looking at frozen animals from the past) to solve the mystery.

1. The Suspect: The Northern Red-backed Vole

The scientists suspected that small, furry animals were the "carriers" of the virus, much like how mosquitoes carry malaria. They focused on a specific suspect: the Northern Red-backed Vole (a tiny rodent that looks a bit like a mouse but has a reddish stripe on its back).

• The Present-Day Hunt: In 2021 and 2024, the team set up traps near where humans had gotten sick. They caught hundreds of small animals.
• The Smoking Gun: When they tested the animals, they found that the voles were the only ones carrying the actual virus DNA and, in one case, a live, growing virus. It was like finding the thief holding the stolen goods.
• The "House Mouse" Clue: They also found antibodies (signs of past infection) in a House Mouse. This is interesting because mice live in our homes. It suggests that if a mouse gets the virus, it could potentially bring it right into your kitchen, making the risk of infection higher for people living in the area.

2. The Time Machine: The Museum Collection

Here is where the story gets really cool. The scientists didn't just look at animals caught now; they went to the University of Alaska Museum.

Think of the museum as a time capsule. They had frozen tissue samples from animals caught decades ago, sitting in liquid nitrogen like time travelers waiting to be woken up.

• The Discovery: They tested these "ancient" samples and found the virus in a vole caught in 1998 and 1999.
• The Big Reveal: This was a massive shock. The first human case of this virus wasn't reported until 2015. The museum samples proved the virus had been hiding in Alaska's wildlife for at least 25 years before anyone knew it existed. It wasn't a new invader; it was a long-time resident that had just recently started causing trouble.

3. The "Family Tree" of the Virus

The scientists also looked at the virus's DNA like a family tree.

• They found that the virus infecting people in Fairbanks was slightly different from the virus infecting a person on the Kenai Peninsula (a different part of Alaska).
• This suggests that the virus isn't just one big outbreak spreading from person to person. Instead, it's like a leaky faucet. The virus lives in the wild (the reservoir), and occasionally, it "drips" over into humans (spillover) in different places and at different times. It's not one continuous chain of infection, but several separate accidents.

4. Why This Matters

This study teaches us three important lessons:

1. Museums are Goldmines: Old museum collections aren't just for looking at; they are powerful tools for tracking diseases. They can tell us how long a virus has been around and where it used to live, which is impossible to know just by looking at current patients.
2. The Virus is Older Than We Thought: Borealpox has been in Alaska for decades. This changes how we think about its origin; it wasn't brought in from outside recently, it was already there, waiting.
3. The Vole is the Key: The Northern Red-backed Vole is likely the main "home" for this virus. Since these voles live in forests but also wander near human houses, they are the bridge between the wild and us.

In a nutshell: Scientists used modern traps and ancient museum jars to prove that a scary virus has been hiding in Alaskan voles for at least 25 years. It's not a new invader, but an old neighbor that occasionally wanders into human territory, causing illness. By understanding this history, we can better protect people in the future.

Technical Summary

1. Problem Statement

Borealpox virus (BRPV), formerly known as Alaskapox, is an orthopoxvirus (OPXV) that has caused seven human infections in Alaska since 2015, including a fatal case in 2023. Despite these spillover events, the natural reservoir host and the temporal history of the virus in North America remained unknown.

• Key Uncertainties:
  • Which wild small mammal species maintains the virus?
  • Is the virus endemic to Alaska or a recent introduction?
  • What is the geographic range of the virus beyond reported human cases (Fairbanks and Kenai Peninsula)?
• Context: Previous investigations suggested small mammals (voles, shrews, squirrels) as potential vectors, but earlier sampling yielded limited positive results. The genetic relationship of BRPV to "Old World" orthopoxviruses suggested a long evolutionary history, yet its presence in Alaska prior to 2015 was unproven.

2. Methodology

The study employed a dual approach combining contemporary field surveillance with retrospective analysis of historical museum collections.

A. Field-Based Sampling (2021 & 2024)

• Locations:
  • Fairbanks/North Pole (Sept 2021): 9 sites near human case residences and family homes.
  • Kenai Peninsula (Aug 2024): 3 sites near the 2023 fatal case location.
• Specimens: 337 wild small mammals trapped using Sherman, Tomahawk, and pitfall traps.
  • Target Species: Northern Red-backed Voles (Clethrionomys rutilus), shrews (Sorex spp.), squirrels (Tamiasciurus, Glaucomys), and others.
• Diagnostics:
  • Serology: ELISA for anti-OPXV IgG antibodies.
  • Molecular: Real-time PCR for OPXV DNA.
  • Virology: Viral isolation attempts on BSC-40 cell lines.
  • Sequencing: Whole-genome sequencing of positive isolates.

B. Museum-Based Retrospective Screening

• Source: Frozen tissue samples from the University of Alaska Museum (UAM) Mammal Collection.
• Specimens: 285 samples collected between 1991 and 2019, focusing on voles, shrews, and squirrels.
• Protocol: Sub-sampling of frozen tissues (liver, mixed tissue) under strict contamination controls (10% bleach, liquid nitrogen handling) followed by OPXV PCR screening.

C. Phylogenetic Analysis

• Genomics: Whole-genome assembly using Illumina MiSeq data.
• Tree Construction: Maximum-likelihood (IQ-TREE) and Bayesian (BEAST) analyses to determine evolutionary relationships between human isolates, contemporary animal isolates, and historical museum isolates.

3. Key Results

A. Contemporary Field Findings (2021 & 2024)

• Seroprevalence: Evidence of past OPXV exposure was found in five species.
  • C. rutilus: 13.6% (Fairbanks) and 8.4% (Kenai) IgG positive.
  • Other positives: American Red Squirrels, Northern Flying Squirrels, a shrew, and a House Mouse (Mus musculus).
• Viral Detection:
  • PCR: Only C. rutilus tested positive for OPXV DNA (7/147 in 2021; 1/83 in 2024).
  • Viable Virus: Successfully isolated from one C. rutilus collected in Kenai (2024).
  • Sequencing: Confirmed as BRPV. No visible pox lesions were observed on PCR-positive animals.

B. Historical Museum Findings

• Detection: 4 out of 201 C. rutilus museum specimens were PCR positive for OPXV DNA.
• Viable Virus: Viable BRPV was successfully isolated from two samples (UAM 51567 and a mixed tissue sample from the same specimen).
• Timeline: These positive specimens were collected in 1998 and 1999 in Denali National Park and Preserve, 17 years before the first reported human case (2015) and 25 years prior to the study's publication.

C. Phylogenetic Insights

• Clade Structure:
  • Fairbanks Clade: Includes human cases (2015–2023), contemporary C. rutilus (2020–2021), and the 1998 museum isolate. This suggests the Fairbanks virus lineage has been circulating for decades.
  • Kenai Clade: Distinct from the Fairbanks clade, containing the 2023 human case and 2024 Kenai C. rutilus. This indicates a separate spillover event or geographic genetic structuring.
• Spillover Dynamics: The lack of monophyly in human-derived sequences supports multiple independent spillover events from a wildlife reservoir rather than sustained human-to-human transmission.

4. Key Contributions

1. Identification of Reservoir: Strongly implicates the Northern Red-backed Vole (Clethrionomys rutilus) as the primary reservoir host for BRPV, capable of maintaining the virus and facilitating spillover.
2. Temporal Extension: Proves BRPV has been endemic in Alaskan wildlife for at least 25 years, predating the first human diagnosis by nearly two decades.
3. Museum Utility: Demonstrates the critical value of natural history museum collections in reconstructing the temporal and spatial history of emerging pathogens.
4. Geographic Expansion: Confirms BRPV presence in Denali National Park, expanding the known range beyond the immediate vicinity of human cases.
5. Novel Host Signal: Detected OPXV antibodies in a House Mouse (Mus musculus), suggesting a potential, previously overlooked transmission pathway in domestic settings.

5. Significance

• Public Health Implications: Understanding that BRPV is a long-standing, endemic wildlife virus rather than a recent import changes the risk assessment. It suggests that spillover is likely driven by ecological factors (e.g., climate change, land use, human-wildlife interface) rather than viral introduction.
• Reservoir Management: Identifying C. rutilus as the reservoir allows for targeted surveillance and risk communication, particularly for immunocompromised individuals in forested or rural Alaskan areas.
• Evolutionary Context: The presence of BRPV in Alaska for decades, despite its genetic affinity to Old World orthopoxviruses, suggests a complex biogeographic history that may involve circulation in the broader Arctic region (Russia/Scandinavia) where C. rutilus is also found.
• Methodological Precedent: The study serves as a model for integrating field epidemiology with "paleovirology" (museum screening) to solve the mysteries of emerging zoonoses.

In conclusion, this study establishes Clethrionomys rutilus as the likely reservoir for Borealpox virus, confirms the virus's long-term endemicity in Alaska, and highlights the necessity of expanded surveillance to prevent future spillover events.