Serotyping of foot-and-mouth disease virus using oxford nanopore sequencing

Abstract

Foot-and-mouth disease virus (FMDV), belonging to the family of Picornaviridae, infects mostly cloven-hoofed animals and leads to huge economic losses. Since there is no cross-protection between the seven serotypes of FMDV, effective vaccination relies on the knowledge of the serotype causing the outbreak. The most common methods of serotyping are antigen ELISAs and amplification-based sequencing. Serotype-specific PCR methods exist but have limitations due to emerging mutants within serotypes. Sequencing is a promising technology, but currently suffers from cumbersome procedures and long turnaround times. In this study, we have established a novel sequencing protocol relying on nanopore sequencing and offline BLAST search. The procedure was completed in 5 h including RNA extraction, reverse transcription, second-strand synthesis, barcoding, sequencing and data analysis, which did not require a bioinformatician. In total, 12,193 sequence files were obtained. The offline BLAST search to the P1 region revealed the most successful categorization of the seven FMDV serotypes (specificity: 98.3%) over whole genome (24.8%), P2 (23.6%) and P3 (21.4%). In conclusion, our protocol enables rapid and reliable FMDV serotyping. The whole procedure can be conducted with a mobile suitcase laboratory, which is easy to use at the point of need in endemic countries.

Keywords: Foot and mouth disease virus; MinION; Nanopore sequencing; Oxford nanopore; Serotyping.