Chapter 65 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. Out of 156 Asian elephants born in captivity in North America during the modern era (between 1962 to 2007), about 25% were stillborn and 78 are still alive. However, of 35 deaths from all causes between the ages of 4 months and 15 years, 20 have been confirmed to be associated with EEHV disease. Overall, the disease has affected approximately 20% of all captive-born Asian elephant calves in North American and European zoos, and has been responsible for two thirds of all deaths of juvenile Asian elephants in captivity in North America. EEHV was first identified in 1995 in association with the index case of lethal acute sudden-onset hemorrhagic disease in a young Asian zoo elephant.15 At least 60 cases with an 85% fatality rate have subsequently been identified by histopathologic examination and polymerase chain reaction (PCR) assay in elephant populations across North America and Europe.4,5,11,14,16 Most of these cases have occurred since 1995 and the rest were detected retrospectively from archival pathologic samples dating back to 1978. Most cases (>80%) have occurred in captive-born juvenile Asian elephant (Elephas maximus) calves younger than 8 years old, with a peak in cases between the ages of 1 and 3 years. Several examples of systemic EEHV have also been confirmed in European stillborn fetuses,18 as well as in older and wild-born animals, and there have been several cases in African elephants (Loxodonta africana). The systemic disease attributed to EEHV has a sudden onset and is characterized by subcutaneous edema of the head and proboscis, cyanosis of the tongue, possibly limb stiffness or lameness, decreased white blood cell and platelet counts, and internal hemorrhages. Histologic abnormalities are predominantly localized to the heart, liver, spleen, tongue, and intestinal tract and other major organs and include the appearance of basophilic intranuclear viral inclusion bodies in the microvasculature of these organs.13,17 By electron microscopy, the inclusion bodies have been found to contain viral capsids morphologically consistent with herpes virions. The 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 resulting from endothelial cell damage caused by the presence of the herpesvirus. Nine calves have survived after early aggressive antiherpesvirus treatment with famciclovir or ganciclovir, but these agents were not uniformly successful in preventing deaths. Furthermore, as many as 18 recent deaths of orphan and wild Asian elephant calves in four different countries in Asia have also been attributed to EEHV.12,23
Current knowledge and all data generated to date have suggested that EEHVs are host-specific to elephants. In general, all mammalian species examined have been found to carry ubiquitous and well-adapted herpesviruses that have evolved colinearly along with their natural hosts. Many host species are infected with a number of different virus species from the alpha-, beta-, and gammaherpesvirus subfamilies. Herpesviruses in general usually cause only mild asymptomatic systemic primary infections at a very early age, then persist long term in a quiescent latent form (usually within neurons, lymphocytes, or monocytes), and occasionally reactivate as localized skin or mucosal epithelial lesions with active lytic infection. These lesions shed infectious virions that may be transmitted by cell to cell contact as well as in aerosols and in body fluids. In the occasional situations in which serious disease is encountered, it is usually presumed to involve a virus that has either become rare or was selected for unusual pathogenicity within its own natural hosts, or more likely has recently crossed species or subspecies boundaries. Alternatively, or in addition, it is also possible the host is immunosuppressed or suffering from concurrent infections.
There are at least seven distinct species or subspecies of EEHV that have been identified and are now collectively classified as members of the Proboscivirus genus within the Betaherpesvirinae: EEHV1A, EEHV1B, EEHV2, EEHV3, EEHV4, EEHV5, and EEHV6.6,9,25 Among the most recent 25 North American cases of systemic infection by EEHVs that have been confirmed, only two (the EEHV5 case and one example of EEHV1B) have not been associated with the typical signs and/or pathologic changes associated with acute hemorrhagic disease. None of the EEHVs have yet been grown in cell culture, but partial genomic DNA sequence analysis after PCR assay directly from necropsy tissue has revealed that they are a highly diverged group that evidently evolved separately from all other herpesvirus genera, beginning about 100 million years ago and corresponding to when the ancestors of modern elephants branched off from all other placental mammals. Three of these viruses (EEHV1A, EEHV2, and EEHV3) have also been found as typical localized reactivated infections in epithelial cells of skin, genital lesions, or pulmonary nodules in healthy African elephants, but EEHV has not been found in peripheral blood from healthy carrier elephants.
Preliminary PCR evidence in early studies has suggested that EEHV1, the virus found most commonly in Asian elephant disease cases, may in fact be endemic in healthy African elephants, because it was detected in an outbreak of skin nodules in several young African elephants from among a large group that was imported to Florida from Zimbabwe in the mid-1980s.8,13,14,16 However, that conclusion has yet to be rigorously or unambiguously confirmed by extensive gene subtyping or in additional samples from wild elephants in Africa. It is plausible that an Asian elephant present at the same location could have transmitted EEHV1 virus to the Florida juveniles after arriving in Florida. On the other hand, EEHV2 was similarly implicated as being present in small nodules found in the lungs of healthy wild African elephants that were culled at Kruger National Park in South Africa in the late 1990s.17 These pulmonary nodules were reported to occur in more than 50% of healthy adult free-ranging animals.10 Furthermore, the presence of EEHV3 and EEHV6 in African elephant lung nodules in high abundance has also recently been confirmed. The original hypothesis was that most cases of this disease in juvenile Asian elephants represent primary infection with a virus (EEHV1A) that has evolved naturally in African elephants, but not in Asian elephants. Over the past decade, this result has led to a significant awareness of and changes in the housing and management of the two species in facilities that keep mixed Asian and African herds, but some cases have occurred at facilities that appear never to have housed African elephants. Current serologic evidence implies that at least 8% of the wild-born adult captive Asian elephants in North America are also carriers of EEHV1 who must themselves have survived primary infection (possibly as infants in the wild), but little is known as yet about the EEHV1 serologic status of captive African elephants or of wild elephants in Asia or Africa. Over the last 2 years, necropsy samples from more than 10 cases of sudden death in both orphaned and wild Asian elephant calves in India were tested for the presence of EEHV by PCR assay. We were able to confirm the presence of EEHV1 DNA in frozen samples from eight of those cases. There have also been reports of at least eight other similarly suspected but unconfirmed cases in other parts of India, Thailand, and Sumatra, plus one PCR-confirmed EEHV1 case each in both Thailand and Cambodia.12,23
Despite over 15 years of research, EEHVs have not yet been successfully grown in cell culture, even in primary Asian or African elephant fibroblasts and placental umbilical cord endothelial cells, nor have stable lymphoid cell lines carrying the virus been established. Therefore, all DNA sequence analyses so far have had to be carried out directly from diseased necropsy tissue or from whole-blood DNA samples obtained from animals with acute EEHV-associated disease. Accurate sequence data have been obtained on both DNA strands for between 22 and 38 kb of EEHV DNA from necropsy tissue from each of five different EEHV1A, EEHV1B, and EEHV2 cases using a combination of lambda phage libraries and PCR approaches with redundant homologous primers.17 Initial viral clones in the libraries were detected by hybridization with EEHV POL or TER probes; genome-walking techniques by direct and PCR-based sequencing were then applied. However, both libraries were incomplete because of the size and amount limitations for DNA recovered from necropsy tissue. Analyses of several additional segments of each virus not available from the libraries and of a few small loci in the four most recently discovered EEHV species were accomplished by direct PCR sequencing approaches based on known homologous motifs as data have been accumulated for more EEHV1 and EEHV2 genes. However, of necessity, all the data available are restricted to within the central core herpesvirus gene block covering about 80 kb out of an expected total genome size range of close to 200 kb for each EEHV species.
All herpesviruses may persist in their host in the form of an episome within the nucleus of various cell types. Generally, the natural host range of herpesviruses is restricted to one species, although there are exceptions. During acute systemic disease, EEHV has been found at very high levels in whole-blood samples from all known cases, and this provides the only definitive test available. At necropsy, viral DNA is also found in all major organs tested in which focal hemorrhaging was evident, including heart, liver, spleen, kidney, colon, intestine, tongue, and brain. With the exception of the localized skin and lung nodules in African elephants and one older Asian elephant, EEHV DNA has never been found in the blood of healthy animals nor in any unrelated autopsy tissue samples. Even in the two cases in which no EEHV1 or EEHV2 was initially found, they eventually both proved instead to contain high levels of two other related viruses of this type, EEHV3 and EEHV4.6,9
EEHV DNA has been detected by PCR assay in necropsies or biopsies of two types of localized nodules found on the skin or in the lungs of otherwise asymptomatic African elephants.17 However, in contrast to the acute disease, both EEHV1 in the skin nodules and EEHV2, EEHV3 and EEHV6 in pulmonary nodules were evidently undergoing productive lytic replication in dermal or alveolar epithelial cells, respectively, as judged by the presence of typical nuclear inclusion bodies. EEHV1 infection was also tentatively identified in vestibular lymphoid patches in some female African elephants, but very few such nodules or patches are known to have been observed in Asian elephants. Both the skin and lung nodules could presumably be considered the EEHV equivalents of reactivated herpes simplex lesions.
Most significantly, among 18 EEHV1A and five EEHV1B genomes for which up to 10 kb of DNA sequencing has been carried out, each by multilocus PCR gene subtyping, multiple minor subtype variants have been recognized.25 In essence, modern EEHV1 genomes are made up of different scrambled combinations of pieces of two genomes that diverged 10 to 20 million years ago, plus residual pieces of three to five distinct genome variants that diverged approximately 1 million years ago. This is superimposed on a common ancestral background from which all currently known individual isolates within both the EEHV1A and EEHV1B subgroups probably diverged within the past 100,000 years. Therefore, there is no doubt that one or the other elephant species has been the natural host of EEHV1 for at least that length of time, and each subtype cluster pattern is probably representative of the various distinctive herds or subpopulations of elephants—whether Asian or African—from which the individual donor wild-born elephant hosts were imported.
In addition to the novel EEHVs, five very distinct species of highly diverged gammaherpesviruses (or EGHVs) have now also been identified in captive Asian and African elephants.22,25 Unlike the elephant EGHVs, which are periodically shed in oral, eye, and genital secretions, with or without visible lesions, in many captive Asian and African elephants, latent state infections have never been detected with any of the EEHVs by standard diagnostic DNA PCR assay in swabs or blood or autopsy tissues from healthy animals that were not already showing clinical signs of acute hemorrhagic disease. The only exceptions, in which EEHV DNA has been detected in blood of seemingly healthy animals, evidently represent either a transient reactivation event, as in the single case of EEHV5 in an older Asian adult, or a very early-stage primary infection in a herdmate of a parallel symptomatic case involving the identical EEHV1B strain. Unlike the pathogenic Proboscivirus, none of the EGHVs have been associated with any specific disease syndromes.