Each of 5 US-origin serotypes of bluetongue virus (BTV) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a BTV serogroup-specific nested polymerase chain reaction (PCR) method and an embryonating chicken egg (ECE) inoculation procedure. Mean duration of viremia was 100 and 38 days for the PCR and ECE methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of BTV-specific PCR products. This enzyme-linked oligonucleotide sorbent assay (ELOSA) relied on annealing of separate biotinylated and fluoresceinated probes to the amplified BTV nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of PCR products indicated that the ELOSA was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The BTV PCR-ELOSA system represents a more sensitive and expeditious means of diagnosing BTV-induced viremia than does the ECE procedure currently used. The combination of ELOSA with PCR should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

An antigen-capture ELISA was used to detect bluetongue virus (BTV) from blood of infected sheep. A rabbit-origin capture antibody and a mouse-origin detection antibody combined with biotin-avidin amplification were used for the assay. The antigen-capture ELISA could not detect virus directly from the blood of infected sheep because of low virus titer. To enhance detection, virus from infected blood was amplified in cell culture. Virus could then be detected from cell culture supernatant fluids, using the ELISA. This amplification step increased the sensitivity of the assay comparable to that of assays performed in cell culture measuring cytopathic effects. The ELISA procedure was specific for BTV and did not mistakenly identify the antigenically related epizootic hemorrhagic disease virus. The antigen-capture ELISA permitted indirect quantitation and identification of BTV from the blood of infected sheep.