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Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the most highly fatal infectious disease among young Asian elephants. Despite the fact that antiviral therapy has been widely used, its therapeutic outcomes remain uncertain. Additionally, the virus has yet to be successfully cultivated in vitro in the process of develop viral envelope glycoproteins for vaccine design. The present study aims to investigate and evaluate EEHV1A glycoprotein B (gB) antigenic epitopes as potential candidates for further vaccine development. Epitopes of EEHV1A-gB were employed in in silico predictions and designed by using online antigenic predicting tools. Candidate genes were then constructed, transformed and expressed in the E. coli vectors prior to examine their potential for acceleration elephant immune responses in vitro. Elephant peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy juvenile Asian elephants were investigated for their proliferative capability and cytokine responses after being stimulated with EEHV1A-gB epitopes. Exposure of elephant PBMCs to 20 µg/mL of gB for 72 h resulted in a significant proliferation of CD3 + cells when compared with the control group. Furthermore, proliferation of CD3 + cells was associated with a marked up-regulation of cytokine mRNA expression, involving IL-1β, IL-8, IL-12 and IFN-γ. It remains to be determined whether these candidate EEHV1A-gB epitopes could activate immune responses in animal models or elephants in vivo. Our potentially promising results demonstrate a degree of feasibility for the use of these gB epitopes in expanding EEHV vaccine development., Competing Interests: Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Elephant endotheliotropic herpesvirus (EEHV) is the causative agent of EEHV-hemorrhagic disease (EEHV-HD) in elephants worldwide. This disease is highly virulent and a predominant cause of fatalities in young Asian elephants. Rapid diagnosis and aggressive therapies have been determined to be a key strategy in the successful treatment of this disease. Herein, we have developed the immunochromatographic strip test for EEHV detection. Accordingly, 31.2 kDa of partial EEHV DNA polymerase (DNApol) protein was expressed in Escherichia coli and used to generate rabbit polyclonal anti-EEHV DNApol antibodies. These were then used to develop an ICS test for EEHV antigen detection using the double-antibody sandwich colloidal gold method. Anti-EEHV DNApol antibodies conjugated with 40 nm colloidal gold solution were used as a detector, while rabbit anti-EEHV DNApol and goat anti-rabbit IgG antibodies immobilized on the nitrocellulose membrane were used as the test and control lines, respectively. The test had a detection limit of 1.25 × 10 5 viral genome copies (vgc)/mL of EEHV obtained from blood samples. Moreover, no specialized equipment or laboratory infrastructure was required in the administration of this test. This developed ICS test for EEHV antigen detection can be used in field application for the rapid detection of EEHV in resource-limited environments., Competing Interests: Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Elephant endotheliotropic herpesvirus (EEHV)-associated haemorrhagic disease (EEHV-HD) is a leading cause of death in Asian elephant calves across the world. Cases of EEHV-HD have been detected in free-living calves through post-mortem examination (PME) indicating the presence of the virus in the wild. In the absence of a non-invasive sampling method, little research into free-living populations has been possible. This study aimed to provide evidence that faeces can be used as a non-invasive sampling method for the detection of EEHV excretion using quantitative polymerase chain reaction., Methods: Serial saliva swabs and faecal samples were taken from five captive Asian elephants in Thailand over 12 weeks. To ensure the presence of detectable elephant DNA within the sample, qPCR was run for amplification of the Asian elephant tumour necrosis factor (TNF-α) gene, EEHV1 and EEHV4., Results: Of 28 sample pairs, seven saliva samples were positive for EEHV, of which two had paired positive faecal samples., Conclusions: This study presents the first evidence that EEHV is excreted in faeces at detectable levels. This method may in future be used for improved understanding of the epidemiology of EEHV in free-living elephant populations, as well as detection of EEHV excretion in captive herds., (© 2021 British Veterinary Association.)

Background: Elephant endotheliotropic herpesvirus causes a hemorrhagic disease (EEHV-HD) that is a major cause of death in juvenile Asian elephants with EEHV1 and EEHV4 being the most prevalent., Aim: To perform a retrospective clinical data analysis., Methods: Records of a total of 103 cases in Thailand confirmed by polymerase chain reaction (PCR) on blood and/or tissue samples., Results: The severity of clinical signs varied among EEHV subtypes. EEHV1A was the most prevalent with 58%, followed by EEHV4 with 34%, EEHV1B with 5.8% and EEHV1&4 co-infection with 1.9%. Overall case fatality rate was 66%. When compared among subtypes, 100% case fatality rate was associated with EEHV1&4 co-infection, 83% with EEHV1B, 75% with EEHV1A, and the lowest at 40% for EEHV4. Calves 2- to 4-year old were in the highest age risk group and exhibited more severe clinical signs with the highest mortality. Majority of cases were found in weaned or trained claves and higher number of cases were observed in rainy season. A gender predilection could not be demonstrated. Severely affected elephants presented with thrombocytopenia, depletion of monocytes, lymphocytes and heterophils, a monocyte:heterophil (M:H) ratio lower than 2.37, hypoproteinemia (both albumin and globulin), severe grade of heterophil toxicity, and low red blood cell counts and pack cell volumes. Survival was not affected by antiviral drug treatment in the severely compromised animals., Conclusion: Early detection by laboratory testing and aggressive application of therapies comprising of supportive and anti-viral treatment can improve survival outcomes of this disease.

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is an acute fatal disease in elephants. Despite the fact that the underlying pathogenesis of EEHV-HD has been proposed, it remains undetermined as to what mechanisms drive these hemorrhagic and edematous lesions. In the present study, we have investigated and explained the pathogenesis of acute EEHV-HD using blood profiles of EEHV-HD and EEHV-infected cases, hematoxylin and eosin (H&E) stain, special stains, immunohistochemistry, quantitative polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). It was found that EEHV genomes were predominantly detected in various internal organs of EEHV-HD cases. Damage to endothelial cells, vasculitis and vascular thrombosis of the small blood vessels were also predominantly observed. Increases in platelet endothelial cell adhesion molecules-1 (PECAM-1)- and von Willebrand factor (vWF)-immunolabeling positive cells were significantly noticed in injured blood vessels. The expression of pro-inflammatory cytokine mRNA was significantly up-regulated in EEHV-HD cases when compared to EEHV-negative controls. We have hypothesized that this could be attributed to the systemic inflammation and disruption of small blood vessels, followed by the disseminated intravascular coagulopathy that enhanced hemorrhagic and edematous lesions in EEHV-HD cases. Our findings have brought attention to the potential application of effective preventive and therapeutic protocols to treat EEHV infection in Asian elephants.

Elephant endotheliotropic herpesvirus (EEHV) is a major cause of death in Asian elephant (Elephas maximus) calves. A 2-year, 11-month-old female, captive Asian elephant presented with facial edema and a mild fever. Blood samples were collected and showed EEHV1A positivity with a high viral load by real time PCR. Heterophil toxicity also was reported for the first time in this case. The calf was treated orally with acyclovir, 45 mg/kg tid for 28 days, which reduced the EEHV1A viral load to undetectable levels within 9 days and the calf survived. A successful outcome with oral acyclovir administration provides another and affordable option to treat EEHV hemorrhagic disease in Asian elephants, and one that is easier to administer in untrained calves.

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Elephant endotheliotropic herpesvirus (EEHV) infection is a conservation threat to the endangered Asian elephant (Elephas maximus), causing fatal hemorrhagic disease in juvenile elephants throughout the world, including Thailand. This study revealed a subclinical EEHV1 infection rate of 5.5% in healthy captive Asian elephants in Thailand (n = 362). The virus was detected in all age classes above one year old, in both sexes, and across the country - even in facilities with no history of hemorrhagic disease (EEHV HD). Subclinical EEHV infection in Thailand urgently requires proper health management.

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-α and IFN-γ. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants., Competing Interests: The authors have declared that no competing interests exist.

Elephant endotheliotropic herpesvirus (EEHV) is one of the most devastating viral infectious diseases in elephants worldwide. To date, it remains unclear how elephants get infected by the virus, where the virus persists, and what mechanisms drive the pathogenesis of the disease. The present study was aimed to develop an antibody against glycoprotein B (gB) of EEHV, investigate the EEHV tissue tropisms, and provide the possible routes of EEHV transmission in Asian elephants. Samples from elephant organs that had died from EEHV1A and EEHV4 infections, peripheral blood mononuclear cells (PBMC) from EEHV4- and non-EEHV-infected calves were used in this study. The results of western immunoblotting indicated that the antibody can be used for detection of gB antigens in both EEHV1A- and EEHV4-infected samples. Immunohistochemical detection indicated that the EEHV gB antigens were distributed mainly in the epithelial cells of the salivary glands, stomach and intestines. Immunofluorescence test of PBMC for EEHV gB in the EEHV4-infected calf indicated that the virus was observed predominantly in the mononuclear phagocytic cells. The findings in the present study unveil tissue tropisms in the EEHV1A- and EEHV4-infected calves and point out that saliva and intestinal content are likely sources for virus transmission in EEHV-infected Asian elephants.

Elephant endotheliotropic herpesvirus (EEHV) is an infection associated with fatal hemorrhagic disease in young Asian elephants ( Elephas maximus). This brief communication describes the postmortem evaluation of two Asian elephant calves diagnosed with EEHV4 and EEHV1A in conjunction with Clostridium perfringens infection. Case 1 was a 7-mo-old, male captive-born Asian elephant that developed diarrhea and died 2 days after clinical presentation. Examination of the heart, lungs, liver, and spleen revealed predominantly basophilic intranuclear inclusion bodies in the endothelial cells of the blood vessels. Case 2 was a 3-mo-old, female wild-born Asian elephant that showed signs of lethargy, anorexia, and convulsions and died 6 hr after clinical presentation. No intranuclear inclusion bodies were observed. The heart, lung, liver, and spleen of both calves tested positive for EEHV by polymerase chain reaction. Phylogenetic analysis identified EEHV4 and EEHV1A in Case 1 and 2, respectively. Additionally, liver, spleen, and hemorrhagic intestinal tissue samples tested positive for C. perfringens α, β, and ε toxins. This is the first reported case to describe coinfection of EEHV and C. perfringens in Asian elephant calves.

Elephant Endotheliotropic Herpesvirus (EEHV) is emerging as a new threat for elephant conservation, since being identified as the cause of severe, often fatal, haemorrhagic disease in young Asian elephants. To describe positive cases and the molecular relatedness of virus detected in elephants in Thailand, we re-examined all available of EEHV samples occurring in young elephants in Thailand between 2006 and 2014 (n=24). Results indicated 75% (18/24) of suspected cases were positive for EEHV by semi-nested PCR. Further gene analysis identified these positive cases as EEHV1A (72%, 13/18 cases), EEHV1B (11%, 2/18) and EEHV4 (17%, 3/18). This study is the first to phylogenetically analyse and provide an overview of most of the known EEHV cases that have occurred in Thailand. Positive individuals ranged in age from one to nine years, with no sex association detected, and occurred across geographical locations throughout the country. All individuals, except one, were captive-born. No history of direct contact among the cases was recorded, and this together with the fact that various subtype clusters of virus were found, implied that none of the positive cases were epidemiologically related. These results concur with the hypothesis that EEHV1 is likely to be an ancient endogenous pathogen in Asian elephants. It is recommended that active surveillance and routine monitoring for EEHV should be undertaken in all elephant range countries, to gain a better understanding of the epidemiology, transmission and prevention of this disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Elephant endotheliotropic herpesvirus (EEHV) is a type of herpesvirus that causes acute hemorrhagic disease in Asian elephants (Elephas maximus) and is often fatal, especially in calves. This study describes the postmortem evaluation of two captive-born Asian elephants (2 and 3 yr of age, respectively) diagnosed with EEHV in Thailand. Both elephants presented only mild depression, lethargy, and anorexia before death within 24 hr of symptom onset. Necropsies were performed, and tissue samples were tested for EEHV viral presence using polymerase chain reaction. Molecular and phylogenetic evidence illustrated two types of EEHV, which were closely related to EEHV1A in Case 1 and EEHV4 in Case 2. Pathologic findings differed between the cases. More specific organ tropism was found in Case 1, where mainly the cardiovascular system was affected. In contrast, in Case 2, hemorrhages were noted in most organs, including in the gastrointestinal, respiratory, and cardiovascular systems. This report is the first to document EEHV4 in Asia and the second case of this strain to be identified in an elephant worldwide.