Real-Time and Nanoplate-Based Digital PCR Assays for the Detection and Absolute Quantification of Borealpox Virus.

Borealpox virus (Alaskapox virus, BRPV), a neglected zoonotic Orthopoxvirus (OPXV), has been reported only in Alaska, usually causing mild infections. A recent fatal case in the Kenai Peninsula has raised concerns about its public health impact. Like other OPXVs, BRPV diagnosis relies on molecular tools, making species-specific assays essential. In this study, we developed and validated two BRPV-specific molecular assays: a TaqMan-based real-time PCR (rtPCR) and a droplet digital PCR (ddPCR) using the QIAcuity platform. Both target the viral CC-chemokines inhibitor (vCCI) gene, showing high sensitivity (limit of detection ~0.3-0.5 copies/μL), excellent specificity (no cross-reactivity with other OPXVs or rash-causing viruses), and strong reproducibility. While rtPCR is ideal for routine diagnostics, ddPCR offers absolute quantification without standard curves, enhancing the detection of low viral loads. Although the lack of clinical BRPV-positive samples limits full validation, both assays show strong potential for improving BRPV detection, helping to distinguish it from other OPXVs and supporting the early identification of emerging orthopoxvirus threats.