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

• Published in: Journal of virological methods Vol. 195; pp. 205 - 210
• Main Authors: Rathogwa, N.M., Quan, M., Smit, J.Q., Lourens, C., Guthrie, A.J., van Vuuren, M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2014
• Subjects: Animals; Diagnostic assay; DNA Primers - genetics; Equine encephalosis virus; Group-specific; Horse Diseases - diagnosis; Horse Diseases - virology; Horses; Orbivirus; Orbivirus - classification; Orbivirus - genetics; Orbivirus - isolation & purification; Real time RT-PCR; Real-Time Polymerase Chain Reaction - methods; Reoviridae Infections - diagnosis; Reoviridae Infections - veterinary; Reoviridae Infections - virology; Sensitivity and Specificity; Sequence Analysis, DNA; Veterinary Medicine - methods; Viral Proteins - genetics; Virology - methods; VP7; Group-specific; Diagnostic assay; VP7; Real time RT-PCR; Equine encephalosis virus; Orbivirus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.10.018

•The S7 genes of 38 EEV isolates were sequenced.•A real-time RT-PCR assay for equine encephalosis virus was developed.•The 95% limit of detection of the EEV assay was 102.9 TCID50.•The assay was specific for EEV and did not detect other orbiviruses (BTV, AHSV).
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-21cells 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 102.9 TCID50/ml blood (95% confidence interval: 102.7 to 103.3).
The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly.