Development of a universal RT-PCR for amplifying and sequencing the leader and capsid-coding region of foot-and-mouth disease virus.

ABSTRACT

Foot-and-mouth disease (FMD) is a highly infectious viral disease of cloven-hoofed animals with debilitating and devastating consequences for livestock industries throughout the world. Key antigenic determinants of the causative agent, FMD virus (FMDV), reside within the surface-exposed proteins of the viral capsid. Therefore, characterization of the sequence that encodes the capsid (P1) is important for tracking the emergence or spread of FMD and for selection and development of new vaccines. Reliable methods to generate sequence for this region are challenging due to the high inter-serotypic variability between different strains of FMDV. This study describes the development and optimization of a novel, robust and universal RT-PCR method that may be used to amplify and sequence a 3kilobase (kb) fragment encompassing the leader proteinase (L) and capsid-coding portions (P1) of the FMDV genome. This new RT-PCR method was evaluated in two laboratories using RNA extracted from 134 clinical samples collected from different countries and representing a range of topotypes and lineages within each of the seven FMDV serotypes. Sequence analysis assisted in the reiterative design of primers that are suitable for routine sequencing of these RT-PCR fragments. Using this method, sequence analysis was undertaken for 49 FMD viruses collected from outbreaks in the field. This approach provides a robust tool that can be used for rapid antigenic characterization of FMDV and phylogenetic analyses and has utility for inclusion in laboratory response programs as an aid to vaccine matching or selection in the event of FMD outbreaks.