Chapter 42 Elephant Herpesviruses
A newly recognized, often-fatal hemorrhagic disease attributed to elephant endotheliotropic herpesvirus (EEHV) has been found in North America, Europe, the Middle East, and Asia. At least two closely related herpesviruses are associated with the disease; one is fatal for Asian elephants, and the other may be lethal for African elephants.
The disease attributed to EEHV has a sudden onset and is characterized by subcutaneous edema of the head and proboscis, cyanosis of the tongue, decreased white blood cell and platelet counts, and internal hemorrhages. Histologic abnormalities are predominantly localized to the heart, liver, tongue, and intestinal tract and include the appearance of basophilic intranuclear viral inclusion bodies in the microvasculature of these organs. On electron microscopy the inclusion bodies contain viral capsids morphologically consistent with herpes virions. The cell type affected by this disease demonstrates that this virus has a predilection for endothelial cells (endotheliotropism), which is unusual for any of the previously characterized herpesviruses. The high fatality rate is attributed to acute myocardial failure and capillary injury and leakage from endothelial cell damage caused by the presence of the herpesvirus.13
ETIOLOGY
Current knowledge and data generated to date suggest that the EEHVs are host specific for elephants. Deoxyribonucleic acid (DNA) extracted from tissues of most of the deceased Asian elephants tested, as well as in the blood of all the survivors treated with famciclovir during their illness, encoded herpesvirus terminase sequences that had only minor variability at the nucleotide level. Initially, no terminase polymerase chain reaction (PCR) products were obtained from African elephants with endothelial disease. Conversely, DNA polymerase–directed PCR generated products from the African cases only, but not from the Asian elephants with endothelial disease. Once new, specific primers were constructed, PCR products almost identical to each other at the nucleotide level were obtained from the DNA polymerase gene from four Asian elephants. However, sequence comparison of the herpesvirus DNA polymerase regions from the two elephant species showed only a 76% protein identity between the viruses detected in Asian and African elephant cases, with 65% identity at the nucleotide level. This indicates two different species of herpesviruses were present in the elephants.13
Similarly, PCR products from the terminase gene region were also obtained from an African elephant once a second set of specific primers were constructed. Sequence comparison of the terminase gene region from the two elephant species showed 80% identity at the nucleotide level, although in this case the changes were all synonymous, and the encoded proteins showed 100% amino acid identity.13 Control samples that proved negative included heart and liver tissue from 10 Asian and 5 African elephants that died from conditions unrelated to endothelial disease. Additionally, DNA extracted from peripheral blood of more than 100 asymptomatic Asian and 41 African elephants, including herd mates from facilities where herpesvirus deaths had occurred, were all negative for herpesvirus using PCR primer sets specific for both viruses.
Polymerase chain reaction sequencing of DNA extracted from African elephant cutaneous papillomas and vulval lymphoid patches encoded protein sequences identical to those obtained from the Asian elephants with EEHV disease.13 Control samples that proved negative included pustular skin lesions from two African and three Asian elephants, without evidence of inclusion bodies, and suppurative or noninflammatory vulval lesions from one Asian and two African elephants. On the other hand, DNA extracted from pulmonary tissue of wild African elephants with morphologic evidence of herpesvirus contained viral sequences in the DNA polymerase gene region that had 100% protein identity with the herpesvirus that was fatal for two African elephants.
The proteins encoded by the PCR-amplified DNA obtained from each of the Asian and African elephants that were affected with the disease are clearly those of herpesviruses, but they are distinct from any of the currently known herpesviruses. By comparing amino acid sequences that are characteristic for each herpesvirus subfamily, together with the results of a phylogenetic tree analysis, the terminase protein of the elephant herpesviruses shows slightly greater similarity to betaherpesviruses than to alphaherpesviruses or gammaherpesviruses, but it is clearly not that of a cytomegalovirus (CMV). Similarly, the elephant virus DNA polymerase proteins do not fit into any of the herpesvirus subgroups.13 These findings within these two highly conserved herpesvirus gene regions, together with the unique pathogenesis, suggest that the causative agents of elephant endothelial disease are either outliers of mammalian betaherpesviruses or belong in a previously unrecognized subfamily.
After extensive sequencing of lambda libraries constructed from diseased tissue from two Asian and one African elephant, it is clear that the EEHVs are unique. Although most of the conserved genes align with the betaherpesviruses, and EEHVs probably contain some beta-specific genes, namely UL82/83 and UL88 (using the human CMV nomenclature), a thymidine kinase (TK) gene homolog is present in both African and Asian EEHV genomes that is not present in any betaherpesvirus sequenced.11 The compelling part of this finding is not only the presence of the TK gene, but that it is adjacent to genes aligning with betaherpesviruses. Furthermore, the African EEHV genome contains both the large and the small subunits of ribonucleotide reductase (RR).11 No other betaherpesvirus contains both subunits of RR, but alphaherpesviruses and gammaherpesviruses contain both RR1 and RR2.
EPIDEMIOLOGY
All herpesviruses may persist in their host in the form of an episome within the nucleus of various cell types. Probably every vertebrate species has at least one herpesvirus that has evolved with the host for millions of years; humans have at least nine herpesviruses. Generally, the natural host range of a herpesvirus is restricted to one species, although there are exceptions. Transmission from one host species to another may occur; for example, simian herpesvirus B (“B virus”), although innocuous in macaques, may be transmitted to humans and is almost invariably fatal.17,18 Herpesviruses are usually highly adapted to their host and rarely cause a lethal disease, except in very young or immunocompromised individuals. Latent herpesvirus infection may be a reservoir of virus that is shed either frequently in excretions or intermittently in lesions.
Before 1995, there were sporadic reports of herpesviruses in elephants. Proliferative cutaneous lesions were described in a herd of captive African elephants.7 The lesions from several of these elephants were removed and examined histologically and ultrastructurally. There was morphologic evidence of herpesvirus within the epidermal cells of the lesions. Consensus primer PCR combined with sequencing yielded molecular evidence that confirmed the presence of herpesvirus sequences identical to those found in Asian elephants with disseminated EEHV disease.13 This finding suggests that at least some of the Asian elephant deaths were potentially caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants.
Vesicles and plaques in the distal urogenital tract of both Asian and African elephants have also been described.1,10 The lesions have been seen in 62% of captive Asian elephants, 89% of captive African elephants, and 90% of free-ranging African elephants. Histologically, they are composed of reactive lymphoid follicles. Only rarely are intranuclear inclusions seen within the lesions; they are present within dendritic cells scattered in the lymphoid tissue.1 Consensus primer PCR for two of five lesions tested in African elephants again yielded molecular evidence of a herpesvirus identical to the EEHV that causes lethal disease in Asian elephants.13 Similar lesions in Asian elephants have not yielded any herpesvirus sequences by PCR.
The third herpesvirus-associated lesions found in African elephants are lung nodules. The nodules are usually gray and vary from spongy to firm in consistency. They are small, up to 3 cm in cross-sectional diameter, and multiple nodules may be found within the same elephant. The nodules have been seen in about 80% of free-ranging African elephants at Kruger National Park.8 Fourteen lung nodules from 10 African elephants were obtained, and PCR sequencing yielded molecular evidence of the same strain of EEHV that is lethal for African elephants.12 Therefore, African elephants carry at least two herpesviruses: one that may be lethal to Asian elephants and the other that is fatal in young African elephants. The status of the Asian elephant as a carrier for either virus has yet to be determined; in closed Asian herds, however, deaths have been attributed to EEHV with no history of exposure to African elephants.
Two genera of elephants are alive today (Loxodonta and Elephas) and originated from a common ancestor, along with Mammuthus, about 3 million years ago. Loxodonta has recently been separated into two distinct species: L. africana (savannah elephant) and L. cyclotis (forest elephant), based on DNA sequence variation in four nuclear genes.14 All modern elephant species migrated out of Africa, and the two African species are separated by geographic barriers, but rare intermingling and breeding do occur. In the last few hundred years, 3 million years of species separation has been challenged by the advent of zoos and wildlife parks that sometimes house both species in the same enclosure. The data presented may indicate the existence of a herpesvirus that is indigenous to and nonlethal in African elephants (perhaps latent in the lymphoid patches and productive in the papillomas). When this virus is present in captive African elephants and inappropriately cross-infects young Asian elephants, the resulting primary disease is apparently lethal. The status of herpesviruses in wild Asian elephants is currently unknown, but a limited survey of lymph node biopsies of Asian elephants in Thailand failed to yield herpesvirus sequences by PCR using direct primers for EEHV.5
Several closed Asian elephant herds have experienced one or more cases of disseminated EEHV, and there is no history of either direct or indirect exposure to African elephants. Additionally, an Asian elephant in Europe delivered a term stillborn fetus with histologic and molecular evidence of EEHV in the placenta and tissues of the fetus. The dam remained completely healthy during this time. This event represents either primary infection of the dam during her gestation period or reactivation of endogenous EEHV. There have been several cases of stillborn Asian elephant fetuses with EEHV in target organs, so it is likely that the virus is at least contributory to the high stillbirth rate in captivity.2 It is still plausible that the EEHV lethal for Asian elephants originated from the African species and has been transmitted to and circulates within the Asian elephant population in captive settings. Some elephants likely develop asymptomatic infections or simply a mild illness that goes unnoticed, then intermittently shed the virus. Young elephants clearly are more susceptible to severe, disseminated illness because a majority of elephant deaths have occurred between 1 and 2.5 years of age. This pattern of transmission has similarities to human herpesviral disease.
Several adult Asian elephants with EEHV have died, and most had similar histories of being housed in isolation for most of their lives. Once the elephants were moved to a new zoo or another elephant entered their facility, they died of EEHV disease. Primary exposure likely occurred when the elephants were moved, and because the animals were immunologically naive, severe illness and death ensued. The correct conditions for primary exposure to EEHV, if the elephant is to survive or be asymptomatic, are not entirely clear, but with the human betaherpesvirus HHV-6, there is individual variation in clinical signs on primary exposure. Some infants develop only a mild rash, whereas others have severe illnesses, including encephalitis and seizures.3,6,9 Strain and host variation may affect disease outcome. A similar situation might also apply to the disease associated with the elephant herpesviruses.
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