EEHV Information2024-04-10T13:59:54-05:00

Key Points about EEHV:

What is EEHV?2024-02-24T00:42:26-06:00
  • EEHV is short for Elephant Endotheliotropic Herpes Virus. The virus is named this because the virus damages the inside lining of blood vessels, which is called endothelium.
  • EEHV is a herpesvirus that infects only elephants. Other animals and people do not become infected or ill from EEHV.
Is EEHV fatal for elephants?2024-02-24T00:43:13-06:00
  • EEHV can cause rapid, often fatal disease, which is called EEHV Hemorrhagic Disease, in elephants that lack adequate immunity to the virus. Most often, EEHV Hemorrhagic Disease occurs in African elephants under 15 years of age and Asian elephants under 10 years of age.
  • EEHV Hemorrhagic disease is the single largest cause of death in young elephants in North America, Europe, and in Thailand. Other regions are data deficient.
How is EEHV detected and treated?2024-02-24T00:43:43-06:00
  • Early and intensive treatment for EEHV Hemorrhagic disease is an elephant’s best chance for survival. Early detection of changes in white blood cells and large amounts of EEHV virus in a young elephant’s blood, even before the elephant acts ill, facilitates the start of early treatment.
  • EEHV Hemorrhagic disease is treated with antiviral medication and supportive care such as fluids, blood and plasma transfusions, vitamins, and immune modulators. Treating a sick elephant requires experienced staff and many hours daily, including around-the-clock care.
  • Being prepared to treat an elephant that is ill from EEHV Hemorrhagic disease and having trained staff, trained elephants, and all the necessary supplies on hand is important to every institution caring for elephants.
Where is EEHV found?2024-02-24T00:44:09-06:00
  • The EEHV viruses have co-evolved with elephants for millions of years. EEHV1, EEHV4, and EEHV5 are found in Asian elephants, and EEHV2, EEHV3, EEHV6 and EEHV7 are found in African elephants.
  • EEHV infection in elephants is a natural part of elephant biology, and most elephants carry EEHV without becoming ill. This is similar to how many herpesviruses behave in other species, including people.
How is EEHV transmitted?2024-02-24T00:44:39-06:00
  • EEHV infection in elephants is not caused by living under human care or human activity. Elephants transmit EEHV to each other through natural behavior, under all conditions and in all countries where elephants live. EEHV has been detected in elephants living in North America, Europe, in South Africa, Australia, and throughout Asia.
What is being done to stop EEHV elephant deaths?2024-02-24T00:45:07-06:00
  • Major advances in understanding EEHV biology, EEHV Hemorrhagic disease diagnosis and treatment, and immunity to EEHV have been made in the last two decades. These advances were made possible by funding and samples provided by North American and European zoological institutions.
  • Recent advances in the ability to measure antibody levels against EEHV are a new tool to better identify elephants with the lowest immunity, and highest risk of EEHV Hemorrhagic Disease.
Is there an EEHV vaccine?2024-02-27T14:13:57-06:00
  • Scientists are currently working to develop a vaccine for EEHV1 for Asian elephants, which will hopefully prevent deaths and reduce the degree of illness from EEHV1. Vaccines for the other EEHVs will hopefully follow.  This critical research is being supported by funding and samples provided by North American and European zoological institutions and donors who want to help control this virus that is devastating to elephants.

EEHV, or elephant endotheliotropic herpesvirus, is a herpesvirus that infects elephants. Like herpesviruses that infect other animals and humans, EEHV can live in the body without causing symptoms or illness. Healthy elephants live with EEHV and may occasionally have the virus detectable in their blood (viremia), or shed it intermittently in their trunk secretions, saliva, or feces. EEHV can be detected in an elephant’s blood, trunk secretions, saliva or feces by a molecular test called quantitative polymerase chain reaction, or qPCR. Almost every adult elephant monitored for EEHV has been found to shed the virus at one time or another, and it is considered part of their natural biology for elephants to carry and shed EEHV. Elephants often shed more than one type of EEHV virus throughout their lives, and comparing the viruses that are shed from elephants in different herds and regions of the world can help us understand better how the virus is transmitted and how it has evolved over time.

If an elephant with inadequate immunity to EEHV encounters the EEHV virus when other elephants shed it, the elephant can develop a serious illness called EEHV Hemorrhagic Disease or EEHV-HD. EEHV-HD can be life-threatening and requires immediate, intensive treatment (learn more about EEHV-HD treatment below).

EEHV- HD starts with the virus being detected in the blood or viremia. While many healthy elephants may have low levels of viremia without illness, in EEHV-HD, the virus increases rapidly in the blood. It quickly overwhelms the elephant’s defenses, causing widespread internal damage to the elephant. Sometimes, it can be difficult to determine if early viremia is a natural finding in a healthy elephant, or if it is the start of EEHV-HD. In the early stages of EEHV-HD, the elephant may not look sick, and may still act normally. Looking at the elephant’s white blood cells can help us predict if the elephant is in early EEHV-HD or not, though it is not a guarantee. Knowing the individual elephant’s normal white blood cell parameters can help us interpret new results more accurately. If we are unsure if an elephant is developing EEHV-HD, we may start treatment for EEHV-HD just in case, until we have a definitive diagnosis.

When a calf infected with EEHV is unable to control the virus, it can result in a dangerous and often fatal illness known as EEHV hemorrhagic disease (EEHV-HD). This is due to injuries of the blood vessels caused both by the virus directly killing cells and the calf’s own immune response to the infection. The result is uncontrolled bleeding and leakage from blood vessels until the calf succumbs to complete circulatory collapse.

Just as in humans, at initial herpesvirus infection a competent immune system can hold the virus back from systemic infection, resulting in minimal disease and persistent latent lifelong infection. These infections often have mild or no symptoms. However, if this initial infection is not kept in check, systemic disease can result. In elephant calves, as EEHV viral replication ramps up the virus infects a type of white blood cell, monocytes. The virus can damage or destroy these cells, but one of the most important steps in the development of EEHV-HD is that the virus uses these circulating white blood cells to spread through the bloodstream and infect the cells that line blood vessels throughout the body (endothelial cells). These vessel-lining cells are what gives this herpesvirus its name: “endotheliotropic” means “endothelium-targeting.”

EEHV infection of the endothelial cells has widespread consequences. Damage to the endothelium causes platelets to stick to the surface, causing clots to form and obstruct vessels. This causes injury to the tissues and organs those vessels were supplying. Breaking the vessels’ lining layer also causes tiny but widespread hemorrhages, reducing the blood that remains in circulation. Endothelial cells are potent triggers for both the clotting and the inflammatory responses in the body. When these intertwined systems are overactive, it causes positive feedback loops of damage, inflammation, and coagulation throughout the body. Inflamed and injured vessels leak fluid into the surrounding tissue, resulting in edema (swelling). As widespread clotting continues, platelets and clotting factors are used up, and the body can no longer stop bleeding.

Since these processes are occurring beneath the elephant’s thick skin, it is difficult to see the destruction occurring in the tissues. Swelling of the head and tongue may be visible due to edema. A characteristic blue/purple tongue (lingual cyanosis) is a hallmark of EEHV-HD seen in severe cases. It is the result of hemorrhages and edema in the tissue seen through the thin, clear mucosa of the tongue’s surface.

It is important to note that until there is sufficient damage to result in an inflammatory response, the calf may not have symptoms and be behaviorally normal, which is why monitoring the blood is critical to provide early detection. Identification of viremia in a calf who had previously been negative for that subtype of EEHV can give the caretakers the warning they need to prepare to support the calf if serious symptoms develop. Even if virus testing is not rapidly available, decrease in monocytes is detectable through routine blood monitoring (CBC) and may provide an early warning, though it is not specific to EEHV infection. During EEHV-HD, other blood parameters can give important prognostic information. Chief among these is thrombocytopenia, abnormally low numbers of platelets. This indicates the point at which platelets are depleted faster than they can be replaced by the body and that the animal is entering the dangerous later phases of EEHV-HD with widespread clot formation and uncontrolled bleeding.

Many aspects of this disease process are still areas of active research, and some features are still unclear, but there is good evidence that the virus exacerbates the runaway inflammation (the “cytokine storm”). Investigations into the factors that allow the virus to overcome the calf’s immune system at the initial infection are crucial to future prevention of this disease. Meanwhile, ongoing efforts to gain a better understanding of the virus’s complex effects on immune cells may provide new treatment options. Systematic evaluation of treatment protocols is also underway to continue improvement of care during acute EEHV-HD. The ultimate goal of research on this disease process is to improve outcomes for calves at risk of this terrible disease through prevention, rapid detection, and effective treatment.

EEHV-HD is one of the deadliest diseases affecting the elephant population, both in the wild and in human care. As with all herpes viruses, the virus is latent, meaning it hides within the body until something causes it to become an active infection. Animal care teams who work with these majestic animals, get to know their personalities, their preferences, and their moods, so they are usually the first to notice any changes in their behavior. The onset of symptoms can appear vague at first but can quickly progress, so daily evaluations by their animal care team is imperative. Additionally, elephants should be trained to participate in their own medical care. This participation builds a strong relationship between the elephants, their caretakers, and the veterinary team. Training for behaviors, such as voluntary blood draws and hand injections can be lifesaving when the veterinary teams need to administer treatments.

Although the virus was first detected back in 1995, there is still a lot about EEHV-HD that is unknown, and many of the treatment recommendations are based more on firsthand experience than on proven research. There is still a lot that needs to be understood and research is ongoing on multiple fronts. The ultimate hope is a vaccine can be produced to help protect these animals from developing active viral infections. When the virus is active and shedding, it is a contagious risk to other elephants within proximity. Animal care teams should continue to monitor the rest of the herd for abnormal signs.

Depending on the severity of the case and the institution’s protocol, monitoring and treating an active EEHV-HD case may include antivirals, pain relievers (analgesics), fluid administration, antibiotics for secondary infections, whole blood or plasma transfusions, stem cell therapy, and general supportive care with some treatments being given two to three times a day.
As these cases can require intensive care for prolonged periods and supplies and medications may be needed quickly, there are strong relationships between institutions caring for elephants. When one institution is dealing with an EEHV-HD case, other institutions are standing by to provide assistance. Help may come in the form of medications or supplies, blood products, and even personnel. Having relief teams ready to assist might be necessary if prolonged treatments over weeks of care are needed and additional experience and expertise can go a long way.

Anyone who has taken care of a very ill loved one understands the high degree of physical, psychological, and emotional stress the patient and caregivers go through.

“An EEHV case is very taxing on the animal care and veterinary departments when so much time, energy, close monitoring, and attention to detail is needed for this level of intensive care. It’s hard to describe when an elephant succumbs to this horrible virus after you’ve built these incredibly trusting relationships with these elephants and put your everything into these life-saving measures for them. But, we have support in this community to try to help us all get through it. These losses often feel like losing a member of your own family and the toll it often takes on people’s mental health and well-being can be difficult to cope with.”- Katie Pilgrim-Kloppe, Zoological Manager, Rivers Edge, Saint Louis Zoo.

Many zoos and other institutions who elephants have supported their staff to come together with researchers to share their collective expertise and form the EEHV Advisory Group. The EEHV Advisory Group is working to develop newer testing and treatment options in the fight against EEHV-HD. With everyone’s commitment and hard work, these gentle giants will be protected against this disease.

Protection against herpesvirus-associated disease requires induction of an immune response that is comprised of antibodies and a specialized cell type, known as T cells (often referred to as killer T cells). Two studies from 2018 and 2020 (Fuery et al J. Virol 2018, and Fuery et al J. Virol 2020), identified parts of the virus that induce strong T cell and antibody responses in elephants, which are likely to provide protection against disease. Most importantly, antibody tests were generated that distinguished responses against the different EEHV types and significantly advanced our understanding of elephant immune status to EEHV in younger vulnerable calves.

These new antibody tests were used to confirm an earlier study (Nofs et al 2013, Vet Imm Imm) suggesting that elephants transfer abundant antibodies to their young across the placental barrier before birth (Fuery et al J. Virol. 2020). Furthermore, declining levels or lack of detectable (maternal) antibodies to EEHV over time, most likely due to an absence of infection from EEHV, resulted in some elephants with little to no baseline passive immunity against EEHV. Elephants displaying this type of immune profile appear to be especially vulnerable to clinical illness or even death from a primary infection. Similar findings were independently observed in subsequent publications (Hoornweg at al 2021). While these initial studies were conducted in the context of Asian elephant responses to EEHV, another study documented similar findings in African elephants (Pursell et al Micro Spectrum 2021): both maternal placental antibody transfer and significant decline of detectable antibodies against the relevant EEHV species prior to onset of EEHV-associated illness were observed. Thus, advances in EEHV serology testing can provide important information regarding potential vulnerability of elephants for EEHV-associated disease. EEHV serology is now available through the Utrecht University and Baylor College of Medicine:

These findings suggest that at least some of the factors leading to EEHV-Hemorrhagic Disease (EEHV-HD) may be independent of potential intrinsic deficiencies in elephants since EEHV-HD is occurring within different elephant species. One hypothesis that might explain susceptibility to EEHV HD is that the disease is one of altered timing of infection. EEHV should infect calves with abundant maternal antibodies on board within the first one-to-two years of life. However, smaller herd size may be a contributor, resulting in fewer contacts with herd mates and reduced chances for EEHV transmission. For Asian elephants, average herd size in their natural range countries has declined, driven in part by fragmented habitats and may be a contributor to the discovery of EEHV HD cases in wild elephants. One study in support of this idea found abundant high seropositive rates within large elephant populations (Hoorweg et al Trans Emerg Dis 2022).

In addition to adaptive immunity—that which is conferred by antibodies and T cells—a more general immune response known as the innate response may also play a role in either helping drive the induction of an effective adaptive response or in some cases, may produce harmful effects. Preliminary studies have started to examine this in more detail (Landolfi et al Vet Imm Imm 2009, Edwards et al, Animals, 2020), but more work has yet to be done in this area.

Three major groups of findings have provided a groundwork of knowledge for generating a vaccine against EEHV and include: 1) Earlier work surveilling immune responses in elephants to identify prominent EEHV proteins targeted by elephants, 2) in depth understanding of the gene content in the different EEHV types from genome sequencing efforts, and 3) an understanding of successful or emerging vaccines developed for other herpesviruses. From this work at least three major proteins, known as glycoproteins, are likely major targets for the immune system and a vaccine that can elicit a robust response against them may have success in providing protection against severe infection with EEHV. These proteins are glycoproteins B, H, and L (gB, gH, and gL). All herpesviruses encode some form of these glycoproteins, which serve to promote attachment and entry of infectious virus into host cells. Antibodies and T cells reactive against this core virus machinery is hypothesized to induce protective immunity against EEHV. Vaccines containing these EEHV glycoproteins have now undergone testing in preclinical models with promising results (Pursell et al, Plos One 2022, Clinton et al Vaccine 2022, Clinton et al, submitted 2024), supporting their potential use in elephants.

Major gaps in knowledge remaining to be addressed include:

  • What role does the innate immune response play in either helping generate an effective adaptive response or causing immune responses that are harmful?
  • What mechanisms account for the lack of some young elephants to successfully mount an immune response against EEHV?
  • What are the conditions required to propagate EEHV in the laboratory?
  • Can additional tests (e.g., virus neutralization assays) be developed to help evaluate vaccine responses with more precision?
  • Until a vaccine is widely available, can plasma from EEHV experienced elephants be used to provide passive immunity against EEHV in elephants with declining or no detectable antibodies against EEHV?
  • Are any of the current anti-herpesvirus drugs efficacious against EEHV?

Worldwide, elephants are under a continuum of human impact. This continuum ranges from elephants under direct human care to free-ranging elephants who live in large, uncontrolled spaces. These large spaces where free-ranging elephants live are often protected by laws, have boundaries or fences, and are frequently impacted by human activities or management. Research shows us that elephants on all aspects of this continuum are living with EEHV.

EEHV infection and/or EEHV Hemorrhagic Disease have been detected in elephants living under human care in most Asian elephant range countries, including Thailand, Myanmar, Laos, Cambodia, Borneo, Mainland Malaysia, Sumatra, China, Sri Lanka, India, and Nepal. EEHV shedding has been documented in free-ranging elephants in China, and EEHV Hemorrhagic Disease has been documented in free-ranging elephants in India. In South Africa, EEHV was detected in lung nodules of culled free-ranging elephants, and more recently in the blood and respiratory samples of healthy free-ranging elephants in Kruger National Park.

Most of what we know about EEHV is based on information from elephants under human care, including zoos, circuses, sanctuaries, orphanages, and elephant camps worldwide. Detection of EEHV in free-ranging elephants requires analysis of elephant samples by a molecular test called polymerase chain reaction (PCR), and these samples can be collected one of two ways. Non-invasive samples such as feces can be collected from the ground and do not require intervention with the elephant. Collection of blood, saliva, trunk, or fecal samples from free-ranging elephants can also be performed while the elephant is under temporary human intervention such as anesthesia for GPS collaring, relocation, or disease sampling. Both methods of sample collection require heavy investment, and more extensive research on free-ranging elephants has been limited by financial resources and researcher bandwidth. Access to EEHV PCR laboratories or permits that allow shipment of elephant samples to an EEHV PCR laboratory in another country is another limiting step in better understanding EEHV in free-ranging elephants.

There are many threats to elephant populations across the world, including human-elephant conflict, poaching, habitat loss, and extreme weather conditions. Currently, it is likely that death or illness due to EEHV Hemorrhagic Disease is not a leading threat to free-ranging elephant populations. Research shows us that elephant populations that are fragmented or reduced in size may not maintain adequate immunity to EEHV, and elephants born into these smaller herds may be at a higher risk of death due to EEHV Hemorrhagic Disease. As free-ranging elephant herds worldwide continue to be impacted by human activity, and as elephant numbers decrease and herds are further fragmented or isolated, illness or death from EEHV Hemorrhagic disease may become a more significant threat to free-ranging elephants in the years to come.

References for EEHV in free-ranging elephants

  • Zachariah A, Zong JC, Long SY, Latimer EM, Heaggans SY, Richman LK, et al. Fatal herpesvirus hemorrhagic disease in wild and orphan Asian elephants in southern India. J Wildl Dis. 2013;49:381–93.
  • Yang N, Bao M, Zhu B, Shen Q, Guo X, Li W, Tang R, Zhu D, Tang Y, Phalen DN, Zhang L. Elephant Endotheliotropic Herpesvirus 1, 4 and 5 in China: Occurrence in Multiple Sample Types and Implications for Wild and Captive Population Surveillance. Viruses. 2022 Feb 17;14(2):411. PMID: 35216004
  • Lakshmi PS, Karikalan M, Sharma GK, Sharma K, Mohan SC, Kumar KR, Miachieo K, Kumar A, Gupta MK, Verma RK, Sahoo N, Saikumar G, Pawde AM. Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India. Microbial Pathogenesis. 2023 Feb 1;175:105972.
  • Hoornweg TE, Perera VP, Karunarathne RNS, Schaftenaar W, Mahakapuge TAN, Kalupahana AW, Rutten VPMG, de Haan CAM. Young elephants in a large herd maintain high levels of elephant endotheliotropic herpesvirus-specific antibodies and do not succumb to fatal hemorrhagic disease. Transbound Emerg Dis. 2022 Jun 27;69(5):e3379-e3385. PMID: 35757981

Severe Viral Disease Found in Elephant Calves: Worldwide Occurrence.

Infections by a large variety of viral pathogens occur ubiquitously throughout the animal kingdom. Some of them including the group known as Herpesviruses are predominantly quiescent, having evolved specialized mechanisms and adaptations for maintaining inapparent long-term interactions. Therefore, although not unprecedented, it was quite surprising and unexpected when a new type of herpesvirus was first discovered to be the cause of an acute (rapid onset) and frequently lethal hemorrhagic disease (EHD) in young Asian elephants in zoos in both the USA and Europe (Richman LK et al, Science, 1999). Subsequently, numerous cases of the same lethal EHD have also been described in many Asian range countries. Characteristic early symptoms include lethargy, facial edema and tongue cyanosis followed by decreased platelets and acute systemic hemorrhagic disease. Initially, herpesvirus-like nuclear inclusion bodies and virion-like particles were identified by standard microscopic and electron microscopic procedures in damaged vascular endothelial cells from the tongue as well as heart, liver, spleen and many other major internal organs during necropsy of a 16-month-old female Asian elephant calf named Kumari at the National Zoo in Washington. Later high levels of just two small DNA segments of a few hundred base pairs in length from two genes (POL and TER) from a novel type of herpesvirus were identified within both blood samples and necropsy tissues from Kumari and several other severely ill active cases of EHD, as well as in preserved archival tissue samples from other earlier unexplained lethal cases. There was also an earlier reported case of EHD-like viremic disease in a circus in Switzerland (Ossent et al, 1988), but the cause was not identified as EEHV1 until later.

Identification of the Proboscivirus Genus by DNA PCR Tests.

Among the eleven DNA-positive cases identified in the study by Richman et al in Science in 1999 there were ten in all in young Asian elephant calves and one in a one-year-old African elephant (Kijana). Short redundant consensus DNA primers (called pan-POL and pan-TER code-hops probes) were used to identify these resulting DNA fragments as belonging to previously unknown mammalian herpesviruses by a process known as polymerase chain reaction (PCR) assays designed to amplify any unknown POL and TER gene DNA that is closely related to that of known classical mammalian herpesviruses. An important point to note was that the viral POL DNA sequence amplified from the tissues of the single African elephant case was similar to but distinctively diverged from those of the ten Asian elephant cases, which were all almost identical to each other at these loci. These novel viruses subsequently became known as Elephant Endotheliotropic Herpesviruses (EEHV) because of the characteristic nuclear inclusion bodies found within vascular endothelial cells lining the blood vessels where the tissue damage and RBC hemorrhaging occurred. The two viruses were designated as distinct species within the Proboscivirus genus with the Asian elephant version being named EEHV1 and the African elephant version being named EEHV2.

Key Features of Mammalian Herpesvirus Sub-Families.

Herpesviruses are large icosahedral enveloped double-stranded DNA viruses that encode between 87 and 164 proteins depending on which sub-family they belong to. They are found infecting almost all vertebrate and mammalian hosts, but with usually strong species restricted host-ranges. Major features of the group are that they each can employ and switch between two distinct genetic programs referred to as “latent state” versus “lytic cycle” infections. The latter uses their own set of six viral DNA-replication proteins leading to productive replication in the nucleus. They have also evolved the characteristic property of being able to hide in a long-term quiescent latent state and have acquired the ability to largely co-exist in a non-pathogenic relationship with their natural hosts. For example, humans can be infected asymptomatically with up to nine different species of herpesviruses that fall into three highly divergent sub-families known as Alpha, Beta and Gamma herpesviruses. But they do occasionally cause diverse medical conditions including cold sores (HSV1), genital lesions (HSV2), chickenpox (VZV), shingles (VZV), congenital CMV disease, microcephaly (CMV), mononucleosis (EBV), Burkitt’s lymphoma (EBV), nasopharyngeal carcinoma (EBV), Kaposi’s sarcoma (KSHV) and systemic organ transplant or AIDS-associated viremias (CMV, HHV6 and EBV). Latency in Alpha herpesviruses targets post-mitotic neurones (HSV), in Beta herpesviruses it involves monocyte/macrophage lineages and in Gamma herpesviruses either immortalized B-cells (EBV) or transformed vascular endothelial cells (KSHV). The EEHVs are now known to be a large cladal group consisting of seven distinct virus species that fall between the Beta and Gamma herpesviruses in phylogenetic trees, but there is still an ongoing debate about whether they should be considered to be classified as an outlier genus within the Beta herpesvirus sub-family as originally designated by the ICTV or instead form a new fourth or Delta sub-family of mammalian herpesviruses as proposed in two papers and a comment published in J Virol by Richman et al (2014), Zong et al (2014) and Pellet (2014). The two most obvious novel standout feature about all of the EEHV genomes that differ dramatically from the other herpesvirus sub-families are firstly the existence of large inversions of part of the overall core gene order and secondly the presence of 28 separate diverged members of a 7xTM membrane protein family evidently derived from a captured host retinoic acid induced protein 3 (RAIP3) gene, including a subset with orphan vGPCR receptor-like characteristics.

Extensive PCR Gene Subtyping and Whole Genome DNA Sequence Analysis.

A great deal of selected PCR gene locus DNA sequencing data, as well as random Illumina next generation complete genome analysis for a total of 18 separate EEHV species and strains (isolates) now in GenBank has provided evidence for seven distinct EEHV species each of which split further into at least two (A and B) clustered sub-species and sometimes more. However, EEHV1A (with eleven complete genomes now in GenBank) is by far the most prevalent and dangerous pathogen having been detected in at least 90% of the cases studied in North American, Europe and India, with just small numbers of lethal cases associated with both EEHV1B and each of the other EEHV species. Strangely, in addition to EEHV1 there have been many more cases of EEHV4 described in Thailand, whereas conversely no cases of EEHV5 at all have been reported as yet from Thailand. Overall, there have been 40 well-characterized DNA-sequence confirmed cases of EEHV hemorrhagic disease or viremia as well as additional high-level throat wash shedding characterized in detail in North America and at least 44 documented cases in Europe, with now more than 140 DNA-positive cases identified in Asian range countries as well, but still none have been reported in continental Africa.

Multiple Different Versions of EEHV in Asian and African Elephant Hosts.

The ability to cause acute systemic disease in up to 20% of young Asian elephants and especially recently also in up to eight young African elephants in human care in North America is not the kind of outcome that we usually expect to see associated with a herpesvirus when interacting with its own natural host. But the nearly universal occurrence of occasional asymptomatic low-level trunk wash and saliva swab shedding, as well as the high frequency of generic EEHV antibody detection especially in adults, leaves no doubt that Asian elephants (Elephas maximus) are indeed the natural hosts of EEHV1A, EEHV1B, EEHV4A. EEHV4B, EEHV5A and EEHV5B, whereas African elephants (probably including both Loxodonta africana and Loxodonta cyclotis) are the natural hosts of EEHV2A, EEHV2B, EEHV3A, EEHV3B, EEHV6 and EEHV7. Nevertheless, only EEHV7 has not yet been associated with a case of lethal EHD, whereas each of the other five non-EEHV1 species has been associated with just a handful of lethal cases despite their much higher levels of asymptomatic shedding or mild symptoms. It is not altogether clear whether this result represents a difference in pathogenicity or a much lower frequency of occurrence than for EEHV1A.

New Serology Tests show that EHD involves Primary Infection.

The recent exciting application of new serology assays that specifically detect antibodies to just certain species or subtypes of the ORF-Q hypervariable antigenic proteins especially (Feury et al, 2020; Purcell et al, 2021) and perhaps also similarly of antibodies to the gH or gO or gL hypervariable antigenic proteins (Hornweeg et al, 2021; 2022) to supplement the use of older cross-reacting conserved gB antibody assays, has clearly demonstrated that acute EHD occurs predominantly as a primary infection in young elephants that do not have the necessary type of maternal or self-generated antibodies to provide protection against serious EEHV1A-associated disease. Presumably, the young victim with a new uncontrollable primary infection has just received the virus through contact with an adult herdmate undergoing a rare reactivation event that had been triggered by a transient switch from latent to lytic infection or (when two or more cases occur nearly simultaneously at the same facility) by transmission from another young herdmate who is also undergoing a primary infection from the same reactivating source animal.

Apparent Lack of Efficacy by Human Antivirals Targeted to EEHV TK and CPK Enzymes.

Surprisingly, expanded PCR gene DNA sequencing analysis of both EEHV1 and EEHV2 revealed that despite being somewhat more closely related overall to the Roseolovirus genus (HHV6A, HHV6B and HHV7) within the Beta herpesvirus sub-family than to the Alpha or Gamma herpesvirus sub-families, they nevertheless encode their own likely functional thymidine kinase (TK) enzymes. This result initially raised hopes that the EEHVs might therefore be susceptible to human antiviral drugs acyclovir (ACV) and famciclovir (FCV) that selectively target the TK enzymes of HSV and EBV (but are absent from HHV6 and CMV) Indeed, several new cases of calves with proven EEHV systemic viremia (EHD) did survive after treatment with these drugs (Doc and Chandra and later Mali and Obert then Jade and Maliha). However, many other treated cases did not survive and there is no direct evidence that the EEHV TK is actually susceptible to these drugs. Moreover, ganciclovir (GCV), which instead acts through the conserved protein kinase (CPK) enzyme of CMV appears to have just very low-level effectiveness in surrogate in vitro assays. But this is perhaps not surprising because both the EEHV1 TK and CPK proteins are only 25% identical at the amino acid level to those of the human HSV and CMV versions that actively target these antiviral drugs.

Viral Loads, Maternal Antibodies and Future Vaccines.

It is now believed that true “survivors” of proven viremic EHD (whether treated with putative anti-virals or not) do so because of early and high-quality veterinary care and/or that they had naturally self-limiting disease and recovered from “lower” viral loads than those that do not. Furthermore, in several cases they had measurably far less viral DNA in the blood and later in cell-free form in the serum than did otherwise similar lethal cases. There is clear evidence that the peak age for lethal symptomatic EEHV1 HD in Asian elephant calves is between the ages of 2 and 8 years, which would seem to correlate with weaning from the mother’s milk. Therefore, quite plausibly the level of viremia (or viral load) reached in the peripheral blood and the relative survivability from EEHV1 HD infection are probably both related to the precise details of the immune status of the victim (i.e. the presence or absence of maternal antibodies and levels of relevant types of self-antibody developed in response to prior mild infections with the same or other EEHV species or strains. The most obvious current trend in EEHV research seems rightly to be targeted towards far more detailed ability to distinguish, evaluate and measure relevant antibody levels. The ultimate goals will be to define the serological status of all potentially susceptible calves (and their herdmates?) and where necessary to generate some form of preventative or at least moderating protective “vaccine”. Admittedly, with the extraordinary high levels of hypervariability between subtypes of many of the most likely antigenic proteins, even among strains of EEHV1 let alone between EEHV species, this could still prove to be a rather complex problem.

High Level Nucleotide Differences Across EEHV1 Strains.

To illustrate the very high level of genetic divergence seen across intact genomes of different EEHV1 strains/isolates, the VIRIDIC DNA difference analysis tool was used to compare the newly completed 179,847-bp genome of EEHV1A(EP55) from one of three identical lethal cases in Switzerland in 2022 to all other complete EEHV1 genomes entered into the GenBank database. This comparison revealed a 4.6% (9,712-bp) nucleotide differences between the new genome and our prototype EEHV1A(Kimba) genome. The data also showed that the closest match to the new genome among all nine other intact EEHV1A genomes was with EEHV1A(IP43) from India at 2.7% (4,856-bp) divergence, whereas the lowest match was with EEHV1(IP143) also from India that had 7.5% (13,488-bp) divergence. Similarly, the divergence from the single available EEHV1B(Emelia) prototype genome was measured at 8.2% (14,747-bp) and the overall pairwise divergence from the single available orthologous EEHV6(MB1) prototype genome from Loxodonta africana was 26.2% (47,120-bp).

Two Major Branches of the EEHVs: Numerous Highly Variable Viral Proteins and Strains.

Like other herpesviruses, which range in size from 115-kb to 240-kb, the EEHVs also have large genomes of 180 to 206-kb encoding up to 120 distinct proteins (including 17 that are spliced) of which about 45 represent conserved “core” genes common to all other herpesviruses as well, but with another 75 being novel new genes not seen before in any other herpesvirus sub-families. Overall, the Probosciviruses also fall into two highly diverged AT-rich versus GC-rich branches that must have diverged at least 40 million years ago (think mastodon versus elephantid hosts, with each but especially EEHV1 and EEHV3 also having a large subset of over 20 genes and proteins that are highly diverged across independent strains (not just between different EEHV species). Furthermore, these variable genes often split into multiple well-defined clusters or cladal branches that we can assign to and define as distinctive gene-level subtypes A, B, C, D, E and F etc. The most dramatic manifestation of this type of variability within a species is the three linked but non-adjacent chimeric loci (CD-I, CD-II and CD-III) totalling some 13-kb and encompassing 12 genes that each display “genus level” protein divergence. This chimeric 1A versus 1B diaspora was defined in detail for the EEHV1A (Kimba, Raman) sub-species compared to the EEHV1B (Emelia) sub-species in Richman et al (2014) and Wilkie et al (2013). But there is still much more additional overall high-level protein variability, which is unlinked across multiple other non-adjacent variable gene loci among and between independent EEHV1A strains. Current evidence indicates that probably all six other EEHV species have somewhat similar overall patterns of at least a 1A versus 1B-like sub-species chimeric diasporas.

Extraordinary R2-Segment Divergence: Multiple Alternative Gene Cassettes.

Finally, even beyond all that there is another very novel pattern of EEHV1A strain diversity found within the small 10.5-kb R2-segment, which is present in the AT-rich branch genomes only: i.e. EEHV1, EEHV2, EEHV5 and EEHV6, but not in EEHV3, EEHV4 or EEHV7 and that does not occur in any other type of herpesvirus. This involves eight distinct alternative three-gene “cassettes” from among a total of 32 independent EEHV1 strains evaluated inserted at one of three different locations and encoding members of either the vGPCR receptor protein family or the vIgFam membrane immunoglobulin-like domain protein family. These patterns allow us to define separate A, B, C, D, E, F, G and H pattern R2-groups. Thus. all the AT-rich EEHV genomes display three major distinct patterns of intra-species strain divergence, but even just a single type and degree of divergence like this is in fact quite rare among other herpesvirus. The exceptions are the three known well-characterized examples of HHV6A and HHV6B, EBV-A and EBV-B and KSHV-P, KSHV-M and KSHV-N that have linked either adjacent or non-adjacent chimeric segments somewhat similar to those of the EEHV1A versus EEHV1B classic chimeric diaspora that appear to have been derived by recombination with other closely related early hominin viral species (eg a closely related orthologous neanderthal virus). Otherwise, only different human strains of CMV encode about twenty unlinked non-adjacent hypervariable proteins that each split into up into as many as eight highly diverged cladal subtype clusters that resemble those of EEHVs but involve a different subset of genes. Finally, nothing resembling the multiple EEHV alternative “cassettes” of EEHV1 have been described in any other mammalian DNA virus. Not surprisingly the R2-segments of EEHV2A, EEHV5A and EEHV5B exhibit major differences in gene content and gene family organization compared to each other and to the EEHV1 version, but whether that extends to also having multiple alternative cassettes in different strains is not yet clear. Even EEHV6 the orthologous virus to EEHV1 in African elephants has a “mini-R2” locus with a total of just four genes encoding an inactivated E47(vFUT9), a frame-shifted U48(vGPCR7) and just one intact member each of the two membrane protein families E49 and E50(vIgFam1). The presence of between three and six long stretches of alternating C and A dinucleotides which could potentially enhance recombination efficiencies is another unique feature of the R2-segments found only in all EEHV1 strains.

Latency, Cell Trophism and Lytic DNA Replication Origins.

With regard to issues such as cell-type tropism and location or genetic program of latent state infections for any of the seven EEHV species, there is very little information accumulated at present, partly because there are no cell culture or laboratory small animal models of any kind available for either lytic propagation or maintenance of a latent state despite significant attempts by us and others to grow EEHV in either primary Asian or African elephant endothelial cell cultures as well as from lymphocytes or myeloid cells in fresh or cryopreserved viremic blood samples. However, as judged by cells in hemorrhagic necropsy tissue detected by histology and microscopy that have large nuclear inclusion bodies, or a subset of cells in viremic blood that give strong signals by in situ hybridization, both vascular endothelial cells and either macrophages, monocytes or neutrophils seem likely to be sites of full lytic replication. Some kind of infection level (but it is not clear whether this is permissive or non-permissive infection) might also apply for certain gut epithelial cells that show apparent positive signals using viral antigen-antibody staining techniques. The only other indirect evidence along these lines is that, whereas the dissected lung nodules from culled African elephants that were very strongly EEHV2 or EEHV3 viral DNA-positive by PCR techniques, these nodules appear to be composed of almost exclusively proliferating lymphocyte follicles, and we suggest purely by analogy with EBV-immortalized cell types that these might all harbor latent state EEHV genomes. However, one thing that has been deduced in comparison with other herpesvirus sub-families is that the mechanism of the predicted lytic DNA replication origin of AT-rich branch EEHVs closely resembles that of Alpha herpesviruses (HSV1, HSV2 and VZV and also HHV6) using a small dyad symmetry element and multiple sites for the UL9 origin binding protein, rather than the completely different mechanisms used by CMV and EBV, although even there the GC-rich branch EEHVs have changed to a larger more complex version of that structure.

Even after several decades of treating EEHV hemorrhagic disease (EEHV-HD) in Asian elephants, we are continuing to evaluate what drugs, doses, and protocols work best in elephants to try and treat this often-fatal disease. The inability to grow the virus has dampened the ability to verify which drugs would be the most effective to treat the virus, however the provision of advanced supportive care has been found to be crucial in managing cases of EEHV-HD. Treatment protocols applied to EEHV-HD in Asian elephants are being applied to Africa elephants with EEHV-HD, although this is still an area that needs additional research.

Vaccine development has been an area of growing interest, and after dedicated research, a vaccine against EEHV-1A is on the horizon. This viral subtype is the most fatal when EEHV-HD is present, and therefore the primary focus of vaccine development. The vaccine is expected to be administered to elephants within the next 48hrs, with strategic distribution across North America. It is important to remember that the vaccine’s aim is to reduce deaths from EEHV, and not eliminate infection as natural virus transmission and circulation within herds will continue to occur.

Commercial serology testing is available for both Asian and African elephants. The detection of antibodies in elephants is indicative of previous infection and successful immune response and therefore assumed protection against EEHV-HD. Since herpesviruses result in lifelong infections, positive serological status also allows us to speculate which animals may shed which virus in the future. In elephants within the at-risk category, negative serostatus and lack of antibodies to a certain viral subtype, is highly suggestive of lack of exposure and the animal being at risk of infection and possibly EEHV-HD. Serostatus can therefore be used to identify animals at risk of infection, which can be used to determine/ dictate the monitoring protocol of that individual animal. These specific animals could then be monitored more closely during times when increased shedding is expected to occur.

Blood and trunk washes are routinely collected from elephants under human care, or at least in human managed environments. Unfortunately, these samples and not possible to collect from elephants in their native range without anesthesia. Globally, researchers are working to determine which non-invasively collected samples from the natural environment can determine if an animal is shedding EEHV, by testing saliva, feces and even chewed up food items that are then dropped on the floor. This data will allow us to determine how many elephants in their native range are carrying the virus, which is currently likely being strongly underestimated.

Future research priorities will focus on better understanding of EEHV shedding and immunity in wild and captive populations in range countries. This includes making antibody testing more widely available. Currently we don’t know what herd factors promote natural immunity or susceptibility to disease. If or when the virus is finally grown in a laboratory environment, a wide range of diagnostic tests will become available to develop and evaluate treatment and vaccine efficacy. Work has not yet started on developing an African elephant EEHV vaccine. Overall, better understanding of what causes a natural elephant virus to cause severe illness in some individuals, will help prevent more elephants dying from this disease.

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