Development and validation of quantitative real-time polymerase chain reaction assays to detect elephant endotheliotropic herpesviruses-2, 3, 4, 5, and 6

► Real-time PCR assays were developed for EEHV2, 3, 4, 5, and 6. ► The new EEHV assays are sensitive and specific. ► The assays are useful for determining EEHV prevalence in in situ and ex situ elephants. ► The assays will be useful for diagnosing suspected cases of EEHV-associated illness. ► The assays will be useful for managing treatment of elephants with EEHV-induced illness. Elephant endotheliotropic herpesviruses (EEHVs) can cause lethal hemorrhagic disease in both African and Asian elephants. At least seven EEHV types have been described, and sensitive real-time PCR tests have been developed for EEHV1A and 1B, which are associated with the majority of characterized Asian elephant deaths. Despite growing knowledge of the different EEHV types, the prevalence of each type within African and Asian elephants remains to be determined and there is considerable need for diagnostic tests to detect and discriminate between each EEHV species for clinical management of African and Asian elephants that develop illness from one or more of these viruses. To begin to address these issues, we developed real-time PCR assays for EEHV2, 3, 4, 5, and 6. Overall, each assay had robust PCR efficiency, a dynamic linear range over 5log10 concentrations, a limit of detection of 10 copies/test reaction with 100% sensitivity, and low intra- and inter-assay variability. Each assay proved to be specific for the EEHV targets for which it was designed, with the exception of EEHV3 and EEHV4, which was expected because of greater DNA sequence similarity between these two EEHV species than the others. These new tools will be useful for conducting surveys of EEHV prevalence within captive and range country elephants, for diagnostic testing of elephants with suspected EEHV-associated disease, and for managing the treatment of elephants with EEHV-induced illness.