Development of a real time polymerase chain reaction assay for equine encephalosis virus.

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 neutralisation test. These procedures are time-consuming and therefore a more rapid diagnostic assay for EEV that can distinguish EEV from African horse sickness virus (AHSV) infections was developed. The S7 (VP7) gene from 38 EEV isolates representing all seven serotypes was amplified and sequenced. A conserved region at the 5' end of the gene was identified and used to design group-specific EEV primers and a TaqMan(®) MGB™ hydrolysis probe. The efficiency of the EEV real-time RT-PCR assay was 81%. The assay was specific, as it did not detect any of the nine serotypes of AHSV, nor 24 serotypes of bluetongue virus (BTV) and sensitive, with a 95% limit of detection of 10(2.9) TCID50/ml blood (95% confidence interval: 10(2.7) to 10(3.3)). The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly.