Brandolini Martina, Ingletto Ludovica, Dirani Giorgio, Zannoli Silvia, Lapusneanu Andreea Maria, Guerra Massimiliano, De Pascali Alessandra Mistral, Lelli Davide, Defilippo Francesco, Lavazza Antonio, Calzolari Mattia, Dottori Michele, Dionisi Laura, Colosimo Claudia, Gatti Giulia, Marzucco Anna, Montanari Maria Sofia, Grumiro Laura, Cricca Monica, Scagliarini Alessandra, Sambri Vittorio
Unit of Microbiology, The Greater Romagna Area Hub Laboratory, Cesena, Italy.
Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy.
Commun Biol. 2025 May 8;8(1):714. doi: 10.1038/s42003-025-08159-9.
Toscana virus (TOSV), a Phlebovirus transmitted by sandflies, is a leading cause of aseptic meningitis in the Mediterranean region. Despite its clinical significance, underreporting and limited availability of complete genomic data hinder a thorough understanding of its genetic diversity and evolution. This study presents a novel amplicon-based whole-genome sequencing (WGS) method using Illumina library preparation kits and proprietary software to optimize workflows and enhance bioinformatic analyses. Primers targeting TOSV lineage A genomes were designed with PrimalScheme to generate 400 bp amplicons, incorporating degenerate bases to improve coverage. Library preparation utilized Illumina Microbial Amplicon Prep (iMAP) kits, followed by de novo assembly using BaseSpace DRAGEN Targeted Microbial software. The method's sensitivity was tested on viral propagates at various RNA concentrations (10 to 10 copies/μL), demonstrating robust performance at concentrations above 10 copies/μL. Validation on high-titre viral propagates (n = 7), low-titre clinical samples (n = 15), and phlebotomine pools (n = 5) confirmed its reproducibility and ability to comprehensively cover coding regions. Cerebrospinal fluid samples yielded the most consistent results compared to urine and sandfly pools. This innovative WGS approach represents a significant advancement in TOSV genomic surveillance, enabling large-scale studies of its genetic diversity and evolutionary dynamics, which are critical for improving diagnostics and public health strategies.
托斯卡纳病毒（TOSV）是一种由白蛉传播的白蛉病毒，是地中海地区无菌性脑膜炎的主要病因。尽管其具有临床意义，但报告不足以及完整基因组数据的可获取性有限，阻碍了对其遗传多样性和进化的全面了解。本研究提出了一种基于扩增子的新型全基因组测序（WGS）方法，该方法使用Illumina文库制备试剂盒和专有软件来优化工作流程并加强生物信息学分析。使用PrimalScheme设计靶向TOSV A谱系基因组的引物，以生成400 bp的扩增子，并掺入简并碱基以提高覆盖率。文库制备使用Illumina微生物扩增子制备（iMAP）试剂盒，随后使用BaseSpace DRAGEN靶向微生物软件进行从头组装。该方法的灵敏度在不同RNA浓度（10至10拷贝/μL）的病毒繁殖体上进行了测试，结果表明在浓度高于10拷贝/μL时具有强大的性能。对高滴度病毒繁殖体（n = 7）、低滴度临床样本（n = 15）和白蛉样本池（n = 5）进行验证，证实了其可重复性以及全面覆盖编码区域的能力。与尿液和白蛉样本池相比，脑脊液样本产生的结果最为一致。这种创新的WGS方法代表了TOSV基因组监测的重大进展，能够对其遗传多样性和进化动态进行大规模研究，这对于改进诊断和公共卫生策略至关重要。
