Reliable detection, sequencing, and transfection of foot-and-mouth disease virus RNA from badly preserved vesicular epithelium
Epithelium or fluid from vesicular lesions are the preferred samples to confirm foot-and-mouth disease virus (FMDV) infection in livestock. A pH-neutral buffered transport medium is recommended for optimal preservation of epithelial samples, but may not be necessary for all circumstances based on the results of our study. Pieces of epithelium were collected from FMDV-infected cattle (isolates O/FRA/1/2001 and A/IRN/22/2015) and stored at room temperature in sealed tubes without any liquid or preservatives. Using RNA extracted from the severely decayed epithelium up to 3 wk after collection, FMDV was successfully detected by RT-rtPCR, and the viral strain was identified by sequencing of capsid protein VP1. Direct isolation of the virus in cell culture was only possible for vesicular material stored for up to 2–5 d, depending on the serotype, but, for both serotypes, infectious virus was recovered by transfection of RNA extracted from epithelium after 3 wk of storage at room temperature. Specialized transport medium will give optimal results, particularly for low-titer samples, but is not required for the reliable detection and characterization of FMDV in highly positive vesicular epithelium by molecular methods.