BIRD-Seq: B2 Protein Integrated End-to-End Pipeline for dsRNA Detection and Nanopore Sequencing for Virus Monitoring

Double-stranded RNA (dsRNA) is a near-universal hallmark of active viral infection. Despite its role as a pan-viral replication intermediate, dsRNA-centred technologies for virus monitoring remain scarce and largely rely on monoclonal antibody-based approaches that, while highly sensitive, are costly and difficult to engineer, scale, or integrate with downstream assays. Here, we present a modular and antibody-free pipeline for quantitative and qualitative dsRNA analysis built around an engineered B2 protein from Flock House virus with nanomolar-range binding affinity. The pipeline is compatible with absorbance- or luminescence-based measurement formats. In a sandwich assay configuration (Sand-BIRD), sub-ng mL-1 quantification of dsRNA is achieved, comparable to the gold standard J2 monoclonal antibody, directly from crude biological samples without RNA extraction. Sand-BIRD reliably detects viral infection in both plant samples (Tomato bushy stunt virus and Grapevine fanleaf virus) and mosquitoes (West Nile virus and Dengue virus) with commercial-grade reliability. dsRNA eluted from positive samples were further processed directly by Oxford Nanopore direct sequencing, enabling identification of virus species without prior sequence knowledge or total RNA extraction. Together, this work establishes an end-to-end, sequence-agnostic workflow for direct RNA-duplex quantification and sequencing (BIRD-Seq), which has compelling potential for emerging infectious disease surveillance and next-generation point-of-care (PoC) diagnostics.