An improved Real-Time Polymerase Chain Reaction for the Simultaneous Detection of All Serotypes of Epizootic Hemorrhagic Disease Virus

Abstract

Epizootic hemorrhagic disease virus (EHDV) is a significant pathogen of wild and sometimes domestic ungulates worldwide. Rapid and reliable methods for virus detection and identification play an essential part in the control of epizootic hemorrhagic disease (EHD). In the present study, a 1-step real-time polymerase chain reaction (PCR) group-specific assay was developed. The assay detects genome segment 5 (NS1) from all of the 8 serotypes of EHDV. Assay sensitivity was evaluated relative to a conventional gel-based nested PCR using cell culture-derived virus and diagnostic samples from clinically affected white-tailed deer (Odocoileus virginianus). The assay reliably amplified the NS1 gene from any of the EHDV strains tested, including isolates from each of the 8 EHDV serotypes. No cross-reactions were detected when all 24 serotypes of Bluetongue virus, a closely related member of the genus Orbivirus, were tested. A panel of 76 known EHDV-positive clinical samples was used to compare the performance of the assay relative to a previously reported real-time PCR assay. Results indicated that there was no statistically significant difference between the threshold cycle values obtained with both assays. A collection of 178 diagnostic samples submitted for EHD diagnosis was also used for test evaluation. The assay could be applied for rapid detection of EHDV in clinical samples from susceptible ruminants during an outbreak of the disease. In addition, this PCR assay has the benefits of being reliable and simple and could provide a valuable tool for studying the epidemiology of EHDV infection in susceptible ruminants by facilitating the detection of EHDV, regardless of the serotype.