Abstract
The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. The Oxford Nanopore Technologies' MinION portable sequencer has the potential to allow sequencing in remote, decentralised laboratories closer to the outbreak location. In this study, we investigated the utility of the MinION to generate sequence data of sufficient quantity and quality for the characterisation of FMDV serotypes O, A, Asia 1. Prior to sequencing, a universal two-step RT-PCR was used to amplify parts of the 5′UTR, as well as the leader, capsid and parts of the 2A encoding regions of FMDV RNA extracted from three sample matrices: cell culture supernatant, tongue epithelial suspension and oral swabs. The resulting consensus sequences were compared with reference sequences generated on the Illumina MiSeq platform. Consensus sequences with an accuracy of 100% were achieved within 10 and 30 min from the start of the sequencing run when using RNA extracted from cell culture supernatants and tongue epithelial suspensions, respectively. In contrast, sequencing from swabs required up to 2.5 h. Together these results demonstrated that the MinION sequencer can be used to accurately and rapidly characterise serotypes A, O, and Asia 1 of FMDV using amplicons amplified from a variety of different sample matrices.
Highlights
Foot-and-mouth disease virus (FMDV) is a highly infectious, positive sense single-stranded RNA virus belonging to the family Picornaviridae and is the causative agent of foot-andmouth disease (FMD) [1]
The full requirement of DNA needed for MinION sequencing (1 μg) was yielded using template derived from cell culture supernatants and epithelium samples infected with A/TAI and ASIA1/IRN and from one of the two O/UKG epithelium samples (O/UKG-EPI-1)
The extensive diversity within and between FMDV serotypes requires the characterisation of many isolates to carry out a detailed epidemiological analysis and as such can complicate the control of the disease [6]
Summary
Foot-and-mouth disease virus (FMDV) is a highly infectious, positive sense single-stranded RNA virus belonging to the family Picornaviridae and is the causative agent of foot-andmouth disease (FMD) [1]. FMD remains a globally important livestock disease affecting domestic and wild cloven-hoofed animals [2]. FMD remains endemic in parts of Africa, Asia, the Middle East and South America, and periodically causes outbreaks in previously free countries and regions [3]. FMD control programs are expensive and trade restrictions prevent the export of animals or animal products from FMD endemic countries. It is estimated that FMD costs between US$6.5–21 billion per year in endemic regions [4].
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