Shaw Andrew E, Lebani Kebaneilwe, González Gordon Lina, Ihearahu Ugonna E, Wadsworth Jemma, Hicks Hayley M, Polo Noemi, Freimanis Graham, Muhanguzi Dennis, Tennakoon Chandana, Orton Richard J, Knowles Nick J, Di Nardo Antonello, Waters Ryan A, Bronsvoort Barend MdeC, King Donald P
The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK.
School of Life Sciences, Botswana International University of Science and Technology, Palapye, Botswana.
BMC Genomics. 2025 Aug 22;26(1):770. doi: 10.1186/s12864-025-11938-7.
Foot-and-mouth disease virus (FMDV) is capable of causing explosive outbreaks among domestic and wild cloven-hoofed animals. Genomic characterisation of FMDV is a crucial component of disease control enabling accurate tracing of disease outbreaks to be undertaken. Nanopore sequencing is an affordable and accessible form of high-throughput sequencing (HTS) technology. However, most published methods for FMDV only sequence genomic fragments or focus upon specific lineages. In this study, a universal FMDV sequencing protocol was developed alongside a bespoke analytical pipeline to sequence any FMDV genome in the absence of prior knowledge regarding the identity of the serotype or lineage.
Universal multiplex RT-PCRs were used to amplify overlapping tiles encompassing the entire FMDV genome. The PCR products were pooled and subjected to nanopore sequencing using the portable MinION sequencing device. A bioinformatics pipeline was used to assemble genomes based upon blastn and reference assembly.
Iterative changes in primer design and pooling resulted in two panels of primers; one set amplifying twenty short fragments (S_scheme), and another set amplifying six longer fragments (L_scheme). Both approaches were shown to be capable of generating FMDV genomes, however the L_scheme was simpler, more reliable and more cost-effective at generating complete genomes. The final L_scheme protocol was assessed using 30 FMDV isolates representing all the currently circulating lineages of FMDV. As part of the development, we successfully trialled the use of this technology in Uganda, a country endemic for FMD.
The amplification, sequencing and bioinformatics strategy developed here has been assessed using a diverse array of FMDV lineages. Using two multiplex PCR reactions, this approach can successfully generate complete genomes of FMDV in a lineage agnostic fashion. Therefore, the primer sets and approaches described here represent a useful tool for expanding the capacity of laboratories to characterise FMDV at the genomic level.
口蹄疫病毒（FMDV）能够在 domestic 和 wild cloven-hoofed 动物中引发爆发性疫情。FMDV 的基因组特征分析是疾病控制的关键组成部分，有助于准确追踪疫情爆发情况。纳米孔测序是一种经济且易于获取的高通量测序（HTS）技术。然而，大多数已发表的 FMDV 方法仅对基因组片段进行测序，或专注于特定谱系。在本研究中，开发了一种通用的 FMDV 测序方案以及一个定制的分析流程，用于在不了解血清型或谱系身份的情况下对任何 FMDV 基因组进行测序。
使用通用多重 RT-PCR 扩增涵盖整个 FMDV 基因组的重叠片段。将 PCR 产物混合，并使用便携式 MinION 测序设备进行纳米孔测序。使用生物信息学流程基于 blastn 和参考组装来组装基因组。
引物设计和混合的迭代变化产生了两组引物；一组扩增二十个短片段（S 方案），另一组扩增六个较长片段（L 方案）。两种方法都能够生成 FMDV 基因组，但 L 方案在生成完整基因组方面更简单、更可靠且更具成本效益。使用代表 FMDV 当前所有流行谱系的 30 个 FMDV 分离株对最终的 L 方案进行了评估。作为开发的一部分，我们在乌干达成功试用了该技术，乌干达是 FMD 的地方流行国家。
这里开发的扩增、测序和生物信息学策略已使用多种 FMDV 谱系进行了评估。通过两个多重 PCR 反应，该方法能够以不依赖谱系的方式成功生成 FMDV 的完整基因组。因此，这里描述的引物组和方法是扩大实验室在基因组水平上表征 FMDV 能力的有用工具。
