Biology subjects

Monsion, B.

Publications and source records attributed to Monsion, B..

3 recordsLinked to original sources

bioRxiv2026

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. Technology ReadinessBIRD-Seq is an integrated pipeline for agnostic dsRNA detection and sequencing designed for broad-spectrum virus monitoring. A Technology Readiness Level (TRL) 5 under NASAs classification framework has been reached as BIRD-Seq has been validated in laboratory-relevant environments using real-world samples, including virus-infected plants and mosquitoes. The ELISA-based sensing platform employs engineered variants of the B2 protein (from Flock House virus) in a sandwich assay format for dsRNA capture and detection, achieving sensitivity comparable to the gold-standard J2 monoclonal antibody. Unlike traditional antibody-based methods, the B2 protein offers key practical advantages: straightforward production in bacterial expression systems, high versatility, and reduced manufacturing costs, as well as direct compatibility with crude extract monitoring, eliminating the need for RNA extraction. Captured B2/RNA duplexes can then be directly eluted from the ELISA microplates and subjected to downstream nanopore direct RNA sequencing, providing both quantitative and qualitative information on the underlying virus infection, a capacity enabled by the near-universal nature of dsRNA as a pathogen-associated molecular pattern. That said, further validation on large-scale field-collected and clinical samples will be essential before widespread deployment can be envisioned. While the B2 sandwich assay offers favorable cost-efficiency over antibody-based alternatives, the relatively high cost of Oxford Nanopore direct RNA sequencing remains an important economic constraint. Nevertheless, the growing importance of dsRNA detection across virus sensing, mRNA vaccine development, innate immunity research, and human disease diagnostics, combined with the increasing role of portable long-read sequencing in emerging infectious disease (EIDs) surveillance, positions BIRD-Seq as an innovative and competitive diagnostic platform. Highlights- Double-stranded RNA (dsRNA) is one of the critical pathogen-associated molecular patterns for viral invasion in the host. A protein-based sandwich assay for dsRNA detection in crude biological samples with a sub-ng mL-1 order detection limit was developed, achieving similar sensing efficiency in comparison to expensive and proprietary monoclonal antibody-based ELISA methods. - The quantitative detection of RNA duplex is coupled with an Oxford nanopore direct dsRNA sequencing method for virus species identification and qualitative analysis. - This is one of the very first dsRNA-centered end-to-end workflows for virus monitoring and sequencing, validated for both infected plant and animal samples.

bioengineering↗

bioRxiv2026

Aphid stylin cuticular proteins contribute to turnip mosaic virus (Potyvirus) transmission

Hundreds of plant viruses are transmitted by aphid vectors, among which non-circulative ones are acquired and inoculated from one host to another within seconds. These viruses are retained on receptors located at the surface of the cuticle of aphid mouthparts. Members of the Potyviridae family are the most abundant RNA viruses infecting plants, and they cause significant economic losses. Among them, viruses of the Potyvirus genus are transmitted by aphids in a stylet-borne manner. Their receptors in aphid stylets remain poorly characterized. Using turnip mosaic virus (TuMV, Potyvirus rapae) as a model, we developed complementary approaches to investigate potyvirus-aphid interactions in three vector species. Immunofluorescence detection and transmission electron microscopy revealed the presence of TuMV in the distal part of aphid maxillary stylets, both in the food canal and on the acrostyle. This cuticular organ houses Stylin proteins, including Stylin-01, the receptor for the cauliflower mosaic virus (Caulimovirus tesselobrassicae). Using CRISPR-Cas9-edited Stylin-01 mutant lines in the pea aphid, we demonstrated that this protein plays an important role in TuMV transmission. Complementary RNA interference silencing experiments revealed that Stylin-04/04bis also mediate TuMV transmission. Furthermore, our findings reveal that targeting simultaneously Stylin-01 and Stylin-04/04bis more strongly impaired the aphids ability to transmit TuMV, suggesting that virus transmission relies on a multi-component stylin interface rather than a single receptor. In conclusion, these results highlight that in complex interactions between potyviruses and their aphid vectors, Stylin proteins are key actors, underscoring their importance in the transmission of stylet-borne viruses. Author summaryPotyviruses are the largest group of plant-infecting RNA viruses and major threats to crops worldwide. Aphids can transmit them within seconds retaining virus particles on their needle-like mouthparts through specific interactions at the stylet surface. However, the identity of the aphid proteins involved in this process has remained unknown. Using turnip mosaic virus (TuMV), a potyvirus with an exceptionnally broad host ranges, we provide evidence of virus retention at the acrostyle, a specialized cuticular microstructure at the tip of aphid stylets. CRISPR-Cas9 stable aphid mutant lines and RNAi-mediated gene silencing revealed that the acrostyle cuticular proteins Stylin-01 and Stylin04/04bis promote TuMV transmission. These findings uncover previously unknown molecular components of the aphid-potyvirus interface and identify potential targets to disrupt virus-vector interaction and virus transmission.

microbiology↗

bioRxiv2019

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes

Plant RNA viruses form highly organized membrane-bound virus replication complexes (VRCs) to replicate their genome and multiply. This process requires both virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) intermediates of replication that trigger potent antiviral defenses in all eukaryotes. In this work, we describe the use of A. thaliana constitutively expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down viral replicating RNA and associated proteins in planta upon infection with tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a number of host proteins, some of which have previously been involved in virus infection. Among a set of nine selected such host candidate proteins, eight showed dramatic re-localization upon infection, and seven of these co-localized with B2-labeled TRV replication complexes, providing ample validation for the immunoprecipitation results. Infection of A. thaliana T-DNA mutant lines for eight of these factors revealed that genetic knock-out of the Double-stranded RNA-Binding protein 2 (DRB2) leads to increased TRV accumulation. In addition, over-expression of this protein caused a dramatic decrease in the accumulation of four unrelated plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We therefore propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore the proteome of VRCs in planta, allowing the discovery of key players in the viral life cycle. AUTHOR SUMMARYViruses are an important class of pathogens that represent a major problem for human, animal and plant health. They hijack the molecular machinery of host cells to complete their replication cycle, a process frequently associated with the production of double-stranded RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected plants, and successfully isolated viral proteins such as the replicase, which attested to the successful isolation of virus replication complexes (VRCs). More excitingly, a number of host proteins, some of which have previously been involved in virus infection, were also captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed dramatic re-localization to viral factories upon infection, and seven of these co-localized dsRNA-labeled VRCs. Genetic knock-out and over-expression experiments revealed that one of these proteins, A. thaliana DRB2, has a remarkable antiviral effect on four plant RNA viruses belonging to different families, providing ample validation of the potential of this experimental approach in the discovery of novel defense pathways and potential biotech tools to combat virus infections in the field. Being compatible with any plant virus as long as it infects Arabidopsis, we propose our dsRNA-centered strategy to be a novel and robust method to explore the proteome of VRCs in planta.

microbiology↗