Laura K. Richman

Clinico-pathologic Features of Fatal Disease Attributed to New Variants of Endotheliotropic Herpesviruses in Two Asian Elephants (Elephas maximus)

The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.

Detection and Evaluation of Novel Herpesviruses in Routine and Pathological Samples from Asian and African Elephants: Identification of Two New Probosciviruses (EEHV5 and EEHV6) and Two New Gammaherpesviruses (EGHV3B and EGHV5)

Systemic infections with elephant endotheliotropic herpesviruses (EEHV) cause a rapid onset acute hemorrhagic disease with an 85% mortality rate. More than 60 cases have been confirmed worldwide occurring predominantly in juvenile Asian elephants. Originally, three virus types EEHV1A, EEHV1B and EEHV2 were identified, all members of the Proboscivirus genus within the Betaherpesvirinae. However, four elephant gammaherpesviruses (EGHV) have also been found by DNA PCR approaches in eye and genital secretions of asymptomatic animals, and two more versions of the probosciviruses, EEHV3 and EEHV4, were recently detected in acute hemorrhagic disease cases. To ask whether even more species of elephant herpesviruses may exist, we have developed several new diagnostic DNA PCR assays using multiple round primers in the DNA POL region. These have been used routinely for nearly three years to screen samples submitted to the Elephant Herpesvirus Laboratory for diagnosis of possible cases of EEHV disease in blood and necropsy tissue, as well as in biopsies of other suspicious lesions or growths. Several more cases of EEHV1-associated hemorrhagic disease were confirmed, but in addition, we describe here eleven examples of other known and novel herpesviruses detected and evaluated with these reagents. They include the prototypes of four new elephant herpesviruses, two more within the proboscivirus group EEHV5 and EEHV6, plus two more gammaherpesviruses EGHV3B and EGHV5. We also report initial semi-quantitative PCR assays demonstrating very high viral loads in the blood of the EEHV3 and EEHV4-associated hemorrhagic disease cases.

FATAL HERPESVIRUS HEMORRHAGIC DISEASE IN WILD AND ORPHAN ASIAN ELEPHANTS IN SOUTHERN INDIA

Up to 65% of deaths of young Asian elephants (Elephas maximus) between 3 mo and 15 yr of age in Europe and North America over the past 20 yr have been attributed to hemorrhagic disease associated with a novel DNA virus called elephant endotheliotropic herpesvirus (EEHV). To evaluate the potential role of EEHV in suspected cases of a similar lethal acute hemorrhagic disease occurring in southern India, we studied pathologic tissue samples collected from field necropsies. Nine cases among both orphaned camp and wild Asian elephants were identified by diagnostic PCR. These were subjected to detailed gene subtype DNA sequencing at multiple PCR loci, which revealed seven distinct strains of EEHV1A and one of EEHV1B. Two orphan calves that died within 3 days of one another at the same training camp had identical EEHV1A DNA sequences, indicating a common epidemiologic source. However, the high level of EEHV1 subtype genetic diversity found among the other Indian strains matches that among over 30 EEHV1 strains that have been evaluated from Europe and North America. These results argue against the previous suggestions that this is just a disease of captive elephants and that the EEHV1 virus has crossed recently from African elephant (Loxodonta africana) hosts to Asian elephants. Instead, both the virus and the disease are evidently widespread in Asia and, despite the disease severity, Asian elephants appear to be the ancient endogenous hosts of both EEHV1A and EEHV1B.

USE OF FAMCICLOVIR FOR THE TREATMENT OF ENDOTHELIOTROPHIC HERPESVIRUS INFECTIONS IN ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract Two juvenile Asian elephants (Elephas maximus) presented with an acute onset of facial edema and lethargy. Examination of the oral cavity of each animal revealed cyanosis of the tip and distal margins of the tongue suggestive of endothelial inclusion body disease (EIBD) of elephants. Whole-blood samples were obtained, and polymerase chain reaction tests confirmed the presence of elephant herpesvirus. The animals were administered famciclovir (Famvir, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19101, USA), a potent human anti-herpesvirus drug, in the course of their disease, and recovery followed a treatment regime of 3–4 wk. These are the first known cases of elephants surviving EIBD.

Comparative Genome Analysis of Four Elephant Endotheliotropic Herpesviruses, EEHV3, EEHV4, EEHV5, and EEHV6, from Cases of Hemorrhagic Disease or Viremia

ABSTRACT The genomes of three types of novel endotheliotropic herpesviruses (elephant endotheliotropic herpesvirus 1A [EEHV1A], EEHV1B, and EEHV2) associated with lethal hemorrhagic disease in Asian elephants have been previously well characterized and assigned to a new Proboscivirus genus. Here we have generated 112 kb of DNA sequence data from segments of four more types of EEHV by direct targeted PCR from blood samples or necropsy tissue samples from six viremic elephants. Comparative phylogenetic analysis of nearly 30 protein-encoding genes of EEHV5 and EEHV6 show that they diverge uniformly by nearly 20% from their closest relatives, EEHV2 and EEHV1A, respectively, and are likely to have similar overall gene content and genome organization. In contrast, seven EEHV3 and EEHV4 genes analyzed differ from those of all other EEHVs by 37% and have a G+C content of 63% compared to just 42% for the others. Three strains of EEHV5 analyzed clustered into two partially chimeric subgroups EEHV5A and EEHV5B that diverge by 19% within three small noncontiguous segments totaling 6.2 kb. We conclude that all six EEHV types should be designated as independent species within a proposed new fourth Deltaherpesvirinae subfamily of mammalian herpesviruses. These virus types likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals and then evolved into two major branches with high- or low-G+C content about 35 million years ago. Later additional branching events subsequently generated three paired sister taxon lineages of which EEHV1 plus EEHV6, EEHV5 plus EEHV2, and EEHV4 plus EEHV3 may represent Asian and African elephant versions, respectively. IMPORTANCE One of the factors threatening the long-term survival of endangered Asian elephants in both wild range countries and in captive breeding populations in zoos is a highly lethal hemorrhagic herpesvirus disease that has killed at least 70 young Asian elephants worldwide. The genomes of the first three types of EEHVs (or probosciviruses) identified have been partially characterized in the preceding accompanying paper (L. K. Richman, J.-C. Zong, E. M. Latimer, J. Lock, R. C. Fleischer, S. Y. Heaggans, and G. S. Hayward, J. Virol. 88:13523–13546, 2014, http://dx.doi.org/10.1128/JVI.01673-14). Here we have used PCR DNA sequence analysis from multiple segments of DNA amplified directly from blood or necropsy tissue samples of six more selected cases of hemorrhagic disease to partially characterize four other types of EEHVs from either Asian or African elephants. We propose that all six types and two chimeric subtypes of EEHV belong to multiple lineages of both AT-rich and GC-rich branches within a new subfamily to be named the Deltaherpesvirinae, which evolved separately from all other mammalian herpesviruses about100 million years ago.

Estimates of the pharmacokinetics of famciclovir and its active metabolite penciclovir in young Asian elephants (Elephas maximus)

OBJECTIVE To determine plasma pharmacokinetics of penciclovir following oral and rectal administration of famciclovir to young Asian elephants (Elephas maximus). ANIMALS 6 healthy Asian elephants (5 females and 1 male), 4.5 to 9 years old and weighing 1,646 to 2,438 kg. PROCEDURES Famciclovir was administered orally or rectally in accordance with an incomplete crossover design. Three treatment groups, each comprising 4 elephants, received single doses of famciclovir (5 mg/kg, PO, or 5 or 15 mg/kg, rectally); there was a minimum 12-week washout period between subsequent famciclovir administrations. Serial blood samples were collected after each administration. Samples were analyzed for famciclovir and penciclovir with a validated liquid chromatography-mass spectroscopy assay. RESULTS Famciclovir was tolerated well for both routes of administration and underwent complete biotransformation to the active metabolite, penciclovir. Mean maximum plasma concentration of penciclovir was 1.3 μg/mL at 1.1 hours after oral administration of 5 mg/kg. Similar results were detected after rectal administration of 5 mg/kg. Mean maximum plasma concentration was 3.6 μg/mL at 0.66 hours after rectal administration of 15 mg/kg; this concentration was similar to results reported for humans receiving 7 mg/kg orally. CONCLUSIONS AND CLINICAL RELEVANCE Juvenile Asian elephants are susceptible to elephant endotheliotropic herpesvirus. Although most infections are fatal, case reports indicate administration of famciclovir has been associated with survival of 3 elephants. In Asian elephants, a dose of 8 to 15 mg of famciclovir/kg given orally or rectally at least every 8 hours may result in penciclovir concentrations that are considered therapeutic in humans.

Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses (EEHVs), Including EEHV2, EEHV3, EEHV6, and EEHV7, within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants

ABSTRACT 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. IMPORTANCE Acute 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.