Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries. A number of single-gene target amplification assays have been developed for the rapid detection of RVF viral RNA. This paper describes the development of an improved amplification assay that includes two confirmatory target RNA segments (L and M) and a third target gene, NSs, which is deleted in the Clone 13 commercial vaccine and other candidate vaccines. The assay also contains an exogenous RNA control added during the PCR setup for detection of amplification inhibitors. The assay was evaluated initially with samples from experimentally infected animals, after which clinical veterinary and human samples from endemic countries were tested for further evaluation. The assay has a sensitivity range of 66.7-100% and a specificity of 92.0-100% depending on the comparison. The assay has an overall sensitivity of 92.5%, specificity of 95% and a positive predictive value of 98.7%. The single-tube assay provides confirmation of the presence of RVFV RNA for improved confidence in diagnostic results and a "differentiate infected from vaccinated animals" (DIVA)--compatible marker for RVFV NSs--deleted vaccines, which is useful for RVF endemic countries, but especially important in non-endemic countries.

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Abstract. Epizootic hemorrhagic disease virus (EHDV) is a significant pathogen of wild and sometimes domestic ungulates worldwide. Rapid and reliable methods for virus detection and identification play an essential part in the control of epizootic hemorrhagic disease (EHD). In the present study, a 1-step real-time polymerase chain reaction (PCR) group-specific assay was developed. The assay detects genome segment 5 (NS1) from all of the 8 serotypes of EHDV. Assay sensitivity was evaluated relative to a conventional gelbased nested PCR using cell culture-derived virus and diagnostic samples from clinically affected white-tailed deer (Odocoileus virginianus). The assay reliably amplified the NS1 gene from any of the EHDV strains tested, including isolates from each of the 8 EHDV serotypes. No cross-reactions were detected when all 24 serotypes of Bluetongue virus, a closely related member of the genus Orbivirus, were tested. A panel of 76 known EHDVpositive clinical samples was used to compare the performance of the assay relative to a previously reported real-time PCR assay. Results indicated that there was no statistically significant difference between the threshold cycle values obtained with both assays. A collection of 178 diagnostic samples submitted for EHD diagnosis was also used for test evaluation. The assay could be applied for rapid detection of EHDV in clinical samples from susceptible ruminants during an outbreak of the disease. In addition, this PCR assay has the benefits of being reliable and simple and could provide a valuable tool for studying the epidemiology of EHDV infection in susceptible ruminants by facilitating the detection of EHDV, regardless of the serotype.