Fall Mamadou L, Xu Dong, Lemoyne Pierre, Clément Geneviève, Moffett Peter, Ritzenthaler Christophe
Saint-Jean-Sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada.
Département de Biologie, Université de Sherbrooke, Sherbrooke, Qué-bec, Canada.
Mol Ecol Resour. 2025 Oct;25(7):e14111. doi: 10.1111/1755-0998.14111. Epub 2025 May 15.
Viral diseases represent a threat to global food production. Managing the impact of viruses on crop production requires the ability to monitor viruses, study their ecology and anticipate outbreaks. Double-stranded RNA (dsRNA) sequencing is a well-established and reliable method of detecting viruses and studying virome-host interactions and ecology. Compared to total RNA extraction, dsRNA extraction eliminates the majority of host RNAs, improving the recovery of viral RNAs. In this study, we developed and evaluated a novel dsRNA extraction method for high-throughput sequencing (HTS) applications based on the Flock House virus (FHV) B2 protein (B2-based method), and compared its performance with that of established cellulose-based and DRB4-based methods (commercial kit), as well as total RNA extraction techniques. The electrostatic properties of B2 have been instrumental in developing a bead-free and resin-free dsRNA extraction method. The B2-based method demonstrated high viral read recovery, achieving proportions exceeding 20% in most samples, and provided better dsRNA purity with less low weight molecule co-extracted RNA than the DRB4-based method and cellulose-based methods. Despite producing overall fewer total reads than the DRB4-based method, the B2-based enrichment for viral-derived dsRNA was better, with a higher percentage of viral reads, making it effective in virome profiling. Furthermore, it had an excellent detection specificity (0.97) and a good detection sensitivity (0.71), minimising false positives and false negatives. In addition, the B2-based method proved to be highly cost-effective, with a per-reaction cost of $4.47, compared to $35.34 for the DRB4-based method. This method offers a practical solution for laboratories with limited resources or for large-scale sampling for viral ecology studies. Future improvements to the B2-based method should focus on optimising sensitivity to Vitivirus species and developing scalable, automated workflows for high-throughput viral detection.
病毒性疾病对全球粮食生产构成威胁。应对病毒对作物生产的影响需要具备监测病毒、研究其生态学并预测疫情爆发的能力。双链RNA（dsRNA）测序是一种成熟且可靠的检测病毒以及研究病毒组与宿主相互作用和生态学的方法。与总RNA提取相比，dsRNA提取消除了大部分宿主RNA，提高了病毒RNA的回收率。在本研究中，我们开发并评估了一种基于 flock house 病毒（FHV）B2蛋白的用于高通量测序（HTS）应用的新型dsRNA提取方法（基于B2的方法），并将其性能与已有的基于纤维素的方法和基于DRB4的方法（商业试剂盒）以及总RNA提取技术进行了比较。B2的静电特性有助于开发一种无珠和无树脂的dsRNA提取方法。基于B2的方法显示出高病毒读数回收率，在大多数样本中达到超过20%的比例，并且与基于DRB4的方法和基于纤维素的方法相比，提取的dsRNA纯度更高，共提取的低分子量分子RNA更少。尽管基于B2的方法产生的总读数总体上比基于DRB4的方法少，但对病毒衍生dsRNA的富集效果更好，病毒读数的百分比更高，使其在病毒组分析中有效。此外，它具有出色的检测特异性（0.97）和良好的检测灵敏度（0.71），最大限度地减少了假阳性和假阴性。此外，基于B2的方法被证明具有很高的成本效益，每个反应的成本为4.47美元，而基于DRB4的方法为35.34美元。该方法为资源有限的实验室或用于病毒生态学研究的大规模采样提供了一种实用的解决方案。基于B2的方法未来的改进应集中在优化对葡萄病毒属物种的灵敏度以及开发用于高通量病毒检测的可扩展、自动化工作流程上。
