Development and evaluation of a real-time polymerase chain reaction assay for equine encephalosis virus

Abstract

Equine encephalosis virus (EEV) is the cause of equine encephalosis. The disease is similar to mild forms of African horse sickness (AHS) and the two diseases are easily confused. Laboratory identification and serotyping of EEV is based on viral isolation in BHK-21 cells and a viral plaque inhibition neutralization test (Erasmus <i<et al., 1970). These procedures require long durations to confirm results and it was desirable that a rapid diagnostic assay was developed to distinguish EEV from African horse sickness virus (AHSV). A PCR test developed for AHSV (Quan et al., 2008) formed the basis for development of a similar assay for EEV. The aim was to develop and evaluate a real time PCR assay for the detection of EEV in the blood and organs of horses. FastPCR software was used to design primers to amplify and sequence the EEV S7 (VP7) gene. RNA was extracted from EEV tissue culture isolates, representing all seven serotypes, using a MagMaxTM Express Particle Processor and MagMaxTM-96 Total RNA Isolation kits. A one step reverse transcription PCR (RT-PCR) was carried out to amplify the EEV S7 gene using a GeneAmp Gold RNA PCR core kit. Sequence reactions were carried out using a BigDye terminal v3.1 sequencing kit and analyzed with an ABI 3130xl Genetic Analyzer. After sequences alignment using BioEdit software, conserved regions were identified and Primer Express 3.0 software was used to design EEV primers and TaqMan® MGBTM hydrolysis probes for real-time RT-PCR assay. The EEV real-time RT-PCR assay was specific and did not detect AHSV nor bluetongue virus (BTV). The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly. Validation of the assay is the next step in establishing it as a routine diagnostic assay. Copyright