DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE^® IN SITU HYBRIDIZATION

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope^® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.