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...

This epidemiologic study follows a 5-yr-old male African elephant ( Loxodonta africana ) during an episode of hemorrhagic disease (HD) due to elephant endotheliotropic herpesvirus 3B (EEHV3B) utilizing data from complete blood counts, electrophoresis and acute phase protein analysis, and polymerase chain reaction (PCR) of multiple body fluids during and after the clinical episode. The elephant presented with sudden onset of marked lethargy and inappetence followed by hypersalivation, hyperemia of the conjunctivae and focally on the tongue, and swellings on the head and ventrum. A moderate leukocytopenia with band neutrophilia, lymphopenia, monocytopenia, and thrombocytophilia was followed by a rise in all three cell types by day 10. Moderate increases in serum amyloid A and C-reactive protein were noted in the first weeks of illness. Conventional PCR of whole blood yielded a strong positive result for EEHV3B. Quantitative PCR revealed moderate viremia, which slowly returned to undetectable levels by day 35 of treatment. EEHV3B was shed in trunk wash samples starting at day 22 for 10 days at moderate levels, and then at low levels for up to 8.5 mo. All three female herd mates shed low levels of EEHV3B in trunk washes intermittently starting from day 28 of the calf's illness until over 7 mo afterward. The majority of saliva samples from the calf over the 8.5-mo period were also positive for EEHV3B. A subfraction of saliva samples from a female herdmate was positive from days 127-190 following disease onset in the calf. Four elephant gammaherpesviruses were detected sporadically from the calf and female herdmates during this same time period. Treatment was started at the onset of clinical signs and consisted of rectal and oral fluids and oral famciclovir. This is the first case of EEHV3B HD in an elephant species and the first thorough epidemiologic evaluation of EEHV HD in an African elephant.

Genes of the vertebrate major histocompatibility complex (MHC) are crucial to defense against infectious disease, provide an important measure of functional genetic diversity, and have been implicated in mate choice and kin recognition. As a result, MHC loci have been characterized for a number of vertebrate species, especially mammals; however, elephants are a notable exception. Our study is the first to characterize patterns of genetic diversity and natural selection in the elephant MHC. We did so using DNA sequences from a single, expressed DQA locus in elephants. We characterized six alleles in 30 African elephants (Loxodonta africana) and four alleles in three Asian elephants (Elephas maximus). In addition, for two of the African alleles and three of the Asian alleles, we characterized complete coding sequences (exons 1-5) and nearly complete non-coding sequences (introns 2-4) for the class II DQA loci. Compared to DQA in other wild mammals, we found moderate polymorphism and allelic diversity and similar patterns of selection; patterns of non-synonymous and synonymous substitutions were consistent with balancing selection acting on the peptides involved in antigen binding in the second exon. In addition, balancing selection has led to strong trans-species allelism that has maintained multiple allelic lineages across both genera of extant elephants for at least 6 million years. We discuss our results in the context of MHC diversity in other mammals and patterns of evolution in elephants.

We provide novel genomic resources to help understand the genomic traits involved in elephant health and to aid conservation efforts. We sequence 11 elephant genomes (5 African savannah, 6 Asian) from North American zoos, including 9 de novo assemblies. We estimate elephant germline mutation rates and reconstruct demographic histories. Finally, we provide an in-solution capture assay to genotype Asian elephants. This assay is suitable for analyzing degraded museum and non-invasive samples, such as feces and hair. The elephant genomic resources we present here should allow for more detailed and uniform studies in the future to aid elephant conservation efforts and disease research.

Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.