Elephant endotheliotropic herpesviruses (EEHVs) can cause acute hemorrhagic disease with high mortality rates in Asian elephants (Elephas maximus). Recently, a new EEHV type known as EEHV5 has been described, but its prevalence and clinical significance remain unknown. In this report, an outbreak of EEHV5 infection in a herd of captive Asian elephants in a zoo was characterized. In February 2011, a 42-yr-old wild-born female Asian elephant presented with bilaterally swollen temporal glands, oral mucosal hyperemia, vesicles on the tongue, and generalized lethargy. The elephant had a leukopenia and thrombocytopenia. She was treated with flunixin meglumine, famciclovir, and fluids. Clinical signs of illness resolved gradually over 2 wk, and the white blood cell count and platelets rebounded to higher-than-normal values. EEHV5 viremia was detectable starting 1 wk before presentation and peaked at the onset of clinical illness. EEHV5 shedding in trunk secretions peaked after viremia resolved and continued for more than 2 mo. EEHV5 trunk shedding from a female herd mate without any detectable viremia was detected prior to onset of clinical disease in the 42-yr-old elephant, indicating reactivation rather than primary infection in this elephant. Subsequent EEHV5 viremia and trunk shedding was documented in the other five elephants in the herd, who remained asymptomatic, except for 1 day of temporal gland swelling in an otherwise-healthy 1-yr-old calf. Unexpectedly, the two elephants most recently introduced into the herd 40 mo previously shed a distinctive EEHV5 strain from that seen in the other five elephants. This is the first report to document the kinetics of EEHV5 infection in captive Asian elephants and to provide evidence that this virus can cause illness in some animals.

More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. IMPORTANCEAcute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly involves a variety of strains of EEHV1, whose natural host has been unclear. Here, we carried out genotype analyses by partial PCR sequencing of necropsy tissue from five asymptomatic African elephants and identified multiple simultaneous infections by several different EEHV types, including high concentrations in lymphoid lung nodules. Overall, the results provide strong evidence that EEHV2, EEHV3, EEHV6, and EEHV7 represent natural ubiquitous infections in African elephants, whereas Asian elephants harbor EEHV1A, EEHV1B, EEHV4, and EEHV5. Although a single case of fatal cross-species infection by EEHV3 is known, the results do not support the previous concept that highly pathogenic EEHV1A crossed from African to Asian elephants in zoos. E lephant endotheliotropic herpesvirus 1 (EEHV1) is the prototype species of the novel Proboscivirus genus that has evidently evolved separately from all other mammalian herpesviruses over the past 100 million years within the ancestors of modern elephants (1-5). Small 250-bp terminase U60(TERex3) and DNA polymerase U38...

Elephant endotheliotropic herpesviruses (EEHV) 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 5 log10 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.

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Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Of the seven known EEHV species, EEHV1 is recognized as the most common cause of hemorrhagic disease among Asian elephants in human care worldwide. Recent data collected from ex situ Asian elephants located in multiple North American and European institutions suggest that subclinical EEHV1 infection is common in this population of elephants. Although fatal EEHV1-associated hemorrhagic disease has been reported in range countries, data are lacking regarding the prevalence of subclinical EEHV infections among in situ Asian elephants. We used previously validated EEHV-specific quantitative real-time PCR assays to detect subclinical EEHV infection in three regionally distinct Asian elephant cohorts, totaling 46 in situ elephants in South India, during October and November 2011. Using DNA prepared from trunk washes, we detected EEHV1, EEHV3/4, and EEHV5 at frequencies of 7, 9, and 20% respectively. None of the trunk washes was positive for EEHV2 or 6. At least one EEHV species was detectable in 35% (16/46) of the samples that were screened. These data suggest that subclinical EEHV infection among in situ Asian elephants occurs and that Asian elephants may be natural hosts for EEHV1, EEHV3 or 4, and EEHV5, but not EEHV2 and EEHV6. The methodology described in this study provides a foundation for further studies to determine prevalences of EEHV infection in Asian elephants throughout the world.