Application of PCR for specific identification of epizootic hemorrhagic disease virus serotype 2

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne double-stranded (ds) RNA virus, is a member of the genus Orbivirus in the family Reoviridae. EHDV infects domestic, captive, and free-ranging ruminants; the whitetailed deer (Odocoileus virginianus) is the species most seriously affected with the disease. The association of EHDV with clinical hemorrhagic disease in cattle is rare. In a previous study, experimental EHDV infection in calves resulted in transient infection that could be detected by virus isolation and serological tests. However, few studies have shown that the virus can also cause bluetongue (BT)-like disease in cattle. 20,21 There are 10 serotypes of EHDV worldwide, 13 but only serotypes 1 and 2 are enzootic in the United States. 17,26,27 Recent epidemiological studies of the disease indicated that EHDV-2 was more prevalent than EHDV-1. Even in the absence of the disease, there is restriction on the international movement of livestock and/or their germplasm from countries suspected to harbor the disease to EHDVfree countries unless the animals are certified free of EHDV infection by serology or virus isolation. Such a restriction could lead to economic losses for EHDV-endemic countries that rely on the sale of livestock and their germplasms for foreign exchange. EHDV isolation procedures presently lack adequate sensitivity. Identification of EHDV field isolates includes direct isolation of the virus in baby hamster kidney (BHK-21) cells or initial inoculation of embryonating chicken eggs (ECE), followed by subsequent passages on cell culture for serotyping. 4,6 Serum neutralization and plaque inhibition tests are commonly used for EHDV serotype-specific identification. However, serology is complicated by cross-reactions within EHDV serogroups and among non-EHDV orbiviruses. The surge of new techniques in cell immunology and molecular biology has made possible the development of improved diagnostic tests. A previous report described a competitive enzyme-linked immunosorbent assay (cELISA) protocol for detection of antibodies to EHDV. However, the cELISA technique required collection of blood samples from animals that have been infected for at least 2 weeks to detect EHDV antibody. cDNA probes were also developed for detection of EHDV serogroup-specific and serotype-specific sequences to serve as efficient alternatives for serotyping. 22,23,30,32 However, these cDNA probes have not proven