Rapid RT-PCR Detection of Epizootic Hemorrhagic Disease Virus Based on NS2 Gene Sequence Analysis of EHDV Serotype 2

A Reverse Transcriptase (RT) Polymerase Chain Reaction (RT-PCR) based assay for rapid detection of Epizootic Hemorrhagic Disease Virus (EHDV) in cell culture was developed and evaluated. A pair of primers (EHD1 and EHD2), selected from gene segment 8 (NS2) of EHDV serotype 2 (Ibaraki strain) was used for RT-PCR amplification. The RT-PCR assay resulted in amplification of a 379 bp PCR product from 100 fg RNAs extracted from EHDV RNAs. RNAs from North American EHDV serotypes 1 and 2 and RNAs from Central African EHDV serotypes 4 and 6 and a number of EHDV field isolates, propagated in cell cultures were detected by this RT-PCR based assay. Amplification products were not detected when the RT-PCR was applied to RNAs from, closely related orbiviruses including; Blue Tongue Virus (BTV) serotypes 2 and 10; Sudanese and South African isolates of Palyam serogroup orbivirus or total nucleic acid extracts from uninfected Vero cells. The described EHDV RT-PCR based assay should provide a simple method for rapid detection of EHDV. The assay should also be recommended for inclusion during epidemiological surveys of the disease in susceptible wild ruminants and domestic livestock in the African continent.