Development of Real-Time RT-PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.