Epizootic hemorrhagic disease: analysis of tissues by amplification and in situ hybridization reveals widespread orbivirus infection at low copy numbers.

A recent outbreak of hemorrhagic fever in wild ruminants in the northwest United States was characterized by rapid onset of fever, followed shortly thereafter by hemorrhage and death. As a result, a confirmed 1,000 white-tailed deer and pronghorn antelope died over the course of 3 months. Lesions were multisystemic and included severe edema, congestion, acute vascular necrosis, and hemorrhage. Animals that died with clinical signs and/or lesions consistent with hemorrhagic fever had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) by radioimmune precipitation but the antibody was limited exclusively to class immunoglobulin M. These findings, indicative of acute infection, were corroborated by the observation that numerous deer were found dead; however, clinically affected deer were rarely seen during the outbreak. Furthermore, only in animals with hemorrhagic lesions was EHDV-2 isolated and/or erythrocyte-associated EHDV-2 RNA detected by serotype-specific reverse transcription (RT)-PCR. By using a novel RT in situ PCR assay, viral nucleic acid was localized to the cytoplasm of large numbers of tissue leukocytes and vascular endothelium in tissues with hemorrhage and to vessels, demonstrating acute intimal and medial necrosis. Because PCR amplification prior to in situ hybridization was essential for detecting EHDV, the virus copy number within individual cells was low, <20 virus copies. These findings suggest that massive covert infection characterized by rapid dissemination of virus facilitates the severe and lethal nature of this disease.