DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

NOVEL DIAGNOSTIC AND THERAPEUTIC APPROACHES TO ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A HEMORRHAGIC DISEASE IN A CAPTIVE JUVENILE ASIAN ELEPHANT (ELEPHAS MAXIMUS).

Novel diagnostic and therapeutic methods were utilized in the successful management of severe elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) in a 1.9-yr-old captive Asian elephant (Elephas maximus). High levels of EEHV1A viremia were detected for 12 d. In addition to established EEHV treatments, therapies included famciclovir-fortified elephant whole blood and plasma, mesenchymal stem cells harvested from elephant umbilical tissue, and aminocaproic acid. Testing conducted to examine the effects of EEHV infection on hemostasis suggested marked intravascular coagulation with decreased plasminogen activity and increased D-dimer concentrations. Thromboelastography was used to assess the efficacy of aminocaproic acid and demonstrated hypofibrinolysis on samples taken after drug administration, as compared with samples from healthy adult Asian elephants. A serological assay for a novel EEHV1A-specific antibody marker (E52) was developed due to lack of seroconversion to a previously established EEHV1A-specific antibody marker (ORFQ) and showed a sustained increase after EEHV-HD illness.

Lethal Hemorrhagic Disease and Clinical Illness Associated with Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, both in captivity and in the wild. Most deaths associated with the virus are caused by two chimeric variants of EEHV1 (EEHV1A and EEHV1B), while two other EEHVs endemic within Asian elephants (EEHV4 and EEHV5) have been recognized but cause death less often. Whether lethal EEHV infections are due to primary infection or reactivation of latent virus remains unknown, and knowledge of the anti-EEHV antibody levels in young elephants is limited. To close these gaps, we sought to develop a serologic assay capable of distinguishing among infections with different EEHVs using a luciferase immunoprecipitation system (LIPS) for antibody profiling and a panel of conserved EEHV recombinant proteins and proteins unique to EEHV1. The results showed that elephants dying from EEHV1 hemorrhagic disease or ill from EEHV infection were seronegative for the EEHV species that caused the disease or illness, indicating that the events were associated with primary infection rather than reactivation of latent virus. We also demonstrated that waning of EEHV1-specific antibodies can occur in the first 2 years of life, when a threshold protective level of antibody may be needed to prevent severe EEHV1-related disease. Use of the LIPS assay to identify putative "diagnostic" proteins would be a valuable asset in determining the EEHV immune status of young elephants and responses to candidate EEHV vaccines in the future.IMPORTANCE Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a longstanding question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of the viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHVs and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.

Asian Elephant T Cell Responses to Elephant Endotheliotropic Herpesvirus.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, an endangered species. One hypothesis to explain this vulnerability of some juvenile elephants is that they fail to mount an effective T cell response to the virus. To our knowledge, there have been no studies of Asian elephant T cell responses to EEHV. To address this deficiency, we validated the gamma interferon (IFN-γ) enzyme-linked immunospot assay for tracking antigen-directed T cell activity by monitoring rabies-specific responses in vaccinated elephants. In addition, we generated monoclonal antibodies to Asian elephant CD4 and CD8 to facilitate phenotypic T cell profiling. Using these tools, we screened healthy elephants with a history of EEHV infection for reactivity against nine EEHV proteins whose counterparts in other herpesviruses are known to induce T cell responses in their natural hosts. We identified glycoprotein B (gB) and the putative regulatory protein E40 as the most immunogenic T cell targets (IFN-γ responses in five of seven elephants), followed by the major capsid protein (IFN-γ responses in three of seven elephants). We also observed that IFN-γ responses were largely from CD4+ T cells. We detected no activity against the predicted major immediate early (E44) and large tegument (E34) proteins, both immunodominant T cell targets in humans latently infected with cytomegalovirus. These studies identified EEHV-specific T cells in Asian elephants for the first time, lending insight into the T cell priming that might be required to protect against EEHV disease, and will guide the design of effective vaccine strategies.IMPORTANCE Endangered Asian elephants are facing many threats, including lethal hemorrhagic disease from elephant endotheliotropic herpesvirus (EEHV). EEHV usually establishes chronic, benign infections in mature Asian elephants but can be lethal to juvenile elephants in captivity and the wild. It is the leading cause of death in captive Asian elephants in North America and Europe. Despite the availability of sensitive tests and protocols for treating EEHV-associated illness, these measures are not always effective. The best line of defense would be a preventative vaccine. We interrogated normal healthy elephants previously infected with EEHV for T cell responses to nine EEHV proteins predicted to induce cellular immune responses. Three proteins elicited IFN-γ responses, suggesting their potential usefulness as vaccine candidates. Our work is the first to describe T cell responses to a member of the proposed fourth subfamily of mammalian herpesviruses, the Deltaherpesvirinae, within a host species in the clade Afrotheria. An EEHV vaccine would greatly contribute to the health care of Asian and African elephants that are also susceptible to this disease.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four ß-strands and an α-helix which form a parallel dimer by interaction of two ß-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

EPIDEMIOLOGIC EVALUATION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 3B INFECTION IN AN AFRICAN ELEPHANT (LOXODONTA AFRICANA).

This epidemiologic study follows a 5-yr-old male African elephant ( Loxodonta africana ) during an episode of hemorrhagic disease (HD) due to elephant endotheliotropic herpesvirus 3B (EEHV3B) utilizing data from complete blood counts, electrophoresis and acute phase protein analysis, and polymerase chain reaction (PCR) of multiple body fluids during and after the clinical episode. The elephant presented with sudden onset of marked lethargy and inappetence followed by hypersalivation, hyperemia of the conjunctivae and focally on the tongue, and swellings on the head and ventrum. A moderate leukocytopenia with band neutrophilia, lymphopenia, monocytopenia, and thrombocytophilia was followed by a rise in all three cell types by day 10. Moderate increases in serum amyloid A and C-reactive protein were noted in the first weeks of illness. Conventional PCR of whole blood yielded a strong positive result for EEHV3B. Quantitative PCR revealed moderate viremia, which slowly returned to undetectable levels by day 35 of treatment. EEHV3B was shed in trunk wash samples starting at day 22 for 10 days at moderate levels, and then at low levels for up to 8.5 mo. All three female herd mates shed low levels of EEHV3B in trunk washes intermittently starting from day 28 of the calf's illness until over 7 mo afterward. The majority of saliva samples from the calf over the 8.5-mo period were also positive for EEHV3B. A subfraction of saliva samples from a female herdmate was positive from days 127-190 following disease onset in the calf. Four elephant gammaherpesviruses were detected sporadically from the calf and female herdmates during this same time period. Treatment was started at the onset of clinical signs and consisted of rectal and oral fluids and oral famciclovir. This is the first case of EEHV3B HD in an elephant species and the first thorough epidemiologic evaluation of EEHV HD in an African elephant.

EBNA2 and Its Coactivator EBNA-LP.

While all herpesviruses can switch between lytic and latent life cycle, which are both driven by specific transcription programs, a unique feature of latent EBV infection is the expression of several distinct and well-defined viral latent transcription programs called latency I, II, and III. Growth transformation of B-cells by EBV in vitro is based on the concerted action of Epstein-Barr virus nuclear antigens (EBNAs) and latent membrane proteins(LMPs). EBV growth-transformed B-cells express a viral transcriptional program, termed latency III, which is characterized by the coexpression of EBNA2 and EBNA-LP with EBNA1, EBNA3A, -3B, and -3C as well as LMP1, LMP2A, and LMP2B. The focus of this review will be to discuss the current understanding of how two of these proteins, EBNA2 and EBNA-LP, contribute to EBV-mediated B-cell growth transformation.

CLINICAL INFECTION OF TWO CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS) WITH ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1B.

The ability of prior infection from one elephant endotheliotropic herpesvirus (EEHV) type to protect against clinical or lethal infection from others remains an important question. This report describes viremia and subsequent shedding of EEHV1B in two juvenile 4-yr-old Asian elephants within 3 wk or 2 mo following significant infections caused by the rarely seen EEHV4. High levels of EEHV1B shedding were detected in the first elephant prior to emergence of infection and viremia in the second animal. The EEHV1B virus associated with both infections was identical to the strain causing infection in two herd mates previously. High EEHV viremia correlated with leukopenia and thrombocytopenia, which was followed by leukocytosis and thrombocytosis when clinical signs started to resolve. The observations from these cases should be beneficial for helping other institutions monitor and treat elephants infected with EEHV1, the most common virus associated with lethal hemorrhagic disease.

CLINICAL INFECTION OF CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS) WITH ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 4.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants. A number of EEHV types and subtypes exist, where most deaths have been caused by EEHV1A and EEHV1B. EEHV4 has been attributed to two deaths, but as both diagnoses were made postmortem, EEHV4 disease has not yet been observed and recorded clinically. In this brief communication, two cases of EEHV4 infection in juvenile elephants at the Houston Zoo are described, where both cases were resolved following intensive treatment and administration of famciclovir. A quantitative real-time polymerase chain reaction detected EEHV4 viremia that correlated with clinical signs. High levels of EEHV4 shedding from trunk wash secretions of the first viremic elephant correlated with subsequent infection of the second elephant with EEHV4. It is hoped that the observations made in these cases--and the successful treatment regimen used--will help other institutions identify and treat EEHV4 infection in the future.

Murine gammaherpesvirus 68 encodes a second PML-modifying protein.

The ORF75c tegument protein of murine gammaherpesvirus 68 (MHV68) promotes the degradation of the antiviral promyelocytic leukemia (PML) protein. Surprisingly, MHV68 expressing a degradation-deficient ORF75c replicated in cell culture and in mice similar to the wild-type virus. However, in cells infected with this mutant virus, PML formed novel track-like structures that are induced by ORF61, the viral ribonucleotide reductase large subunit. These findings may explain why ORF75c mutant viruses unable to degrade PML had no demonstrable phenotype after infection.

A novel antigen capture ELISA for the specific detection of IgG antibodies to elephant endotheliotropic herpes virus.

BACKGROUND: Elephants are classified as critically endangered animals by the International Union for Conservation of Species (IUCN). Elephant endotheliotropic herpesvirus (EEHV) poses a large threat to breeding programs of captive Asian elephants by causing fatal haemorrhagic disease. EEHV infection is detected by PCR in samples from both clinically ill and asymptomatic elephants with an active infection, whereas latent carriers can be distinguished exclusively via serological assays. To date, identification of latent carriers has been challenging, since there are no serological assays capable of detecting seropositive elephants. RESULTS: Here we describe a novel ELISA that specifically detects EEHV antibodies circulating in Asian elephant plasma/serum. Approximately 80 % of PCR positive elephants display EEHV-specific antibodies. Monitoring three Asian elephant herds from European zoos revealed that the serostatus of elephants within a herd varied from non-detectable to high titers. The antibody titers showed typical herpes-like rise-and-fall patterns in time which occur in all seropositive animals in the herd more or less simultaneously. CONCLUSIONS: This study shows that the developed ELISA is suitable to detect antibodies specific to EEHV. It allows study of EEHV seroprevalence in Asian elephants. Results confirm that EEHV prevalence among Asian elephants (whether captive-born or wild-caught) is high.

Promyelocytic leukemia protein modulates establishment and maintenance of latent gammaherpesvirus infection in peritoneal cells.

Promyelocytic leukemia protein (PML) is an essential organizer of PML nuclear bodies (NBs), which carry out a variety of activities, including antiviral functions. Herpesviruses from all subfamilies encode proteins that counteract PML NB-mediated antiviral defenses by multiple mechanisms. However, because of the species specificity of herpesviruses, only a limited number of in vivo studies have been undertaken to investigate the effect of PML or PML NBs on herpesvirus infection. To address this central issue in herpesvirus biology, we studied the course of infection in wild-type and PML⁻/⁻ mice using murine gammaherpesvirus 68 (MHV68), which encodes a tegument protein that induces PML degradation. While acute infection in PML⁻/⁻ mice progressed similarly to that in wild-type mice, the lytic reactivation frequency was higher in peritoneal exudate cells, due to both an increase of MHV68 genome-positive cells and greater reactivation efficiency. We also detected a higher frequency of persistent infection in PML⁻/⁻ peritoneal cells. These findings suggest that the PML protein can repress the establishment or maintenance of gammaherpesvirus latency in vivo. Further use of the PML⁻/⁻ mouse model should aid in dissecting the molecular mechanisms that underlie the role of PML in gammaherpesvirus latency and may yield clues for how PML modulates herpesvirus latency in general.

Acute phase protein expression during elephant endotheliotropic herpesvirus-1 viremia in Asian elephants (Elephas maximus).

Infection of Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus (EEHV) can be associated with rapid, lethal hemorrhagic disease and has been documented in elephant herds in human care and in the wild. Recent reports describe real-time quantitative polymerase chain reaction (qPCR) assays used to monitor clinically ill elephants and also to detect subclinical EEHV1 infection in apparently healthy Asian elephants. Acute phase proteins have been demonstrated to increase with a variety of infectious etiologies in domesticated mammals but have not yet been described in elephants. In addition, the immune response of Asian elephants to EEHV1 infection has not been described. In this study, whole blood and trunk wash samples representing repeated measures from eight elephants were examined for the presence of EEHV1 using a qPCR assay. Elephants were classified into groups, as follows: whole blood negative and positive and trunk wash negative and positive. Serum amyloid A (SAA) and haptoglobin (HP) levels were compared between these groups. A significant difference in SAA was observed with nearly a threefold higher mean value during periods of viremia (P=0.011). Higher values of SAA were associated with >10,000 virus genome copies/ml EEHV1 in whole blood. There were no significant differences in HP levels, although some individual animals did exhibit increased levels with infection. These data indicate that an inflammatory process is stimulated during EEHV1 viremia. Acute phase protein quantitation may aid in monitoring the health status of Asian elephants.

Elephant endotheliotropic herpesvirus 5, a newly recognized elephant herpesvirus associated with clinical and subclinical infections in captive Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesviruses (EEHVs) can cause acute hemorrhagic disease with high mortality rates in Asian elephants (Elephas maximus). Recently, a new EEHV type known as EEHV5 has been described, but its prevalence and clinical significance remain unknown. In this report, an outbreak of EEHV5 infection in a herd of captive Asian elephants in a zoo was characterized. In February 2011, a 42-yr-old wild-born female Asian elephant presented with bilaterally swollen temporal glands, oral mucosal hyperemia, vesicles on the tongue, and generalized lethargy. The elephant had a leukopenia and thrombocytopenia. She was treated with flunixin meglumine, famciclovir, and fluids. Clinical signs of illness resolved gradually over 2 wk, and the white blood cell count and platelets rebounded to higher-than-normal values. EEHV5 viremia was detectable starting 1 wk before presentation and peaked at the onset of clinical illness. EEHV5 shedding in trunk secretions peaked after viremia resolved and continued for more than 2 mo. EEHV5 trunk shedding from a female herd mate without any detectable viremia was detected prior to onset of clinical disease in the 42-yr-old elephant, indicating reactivation rather than primary infection in this elephant. Subsequent EEHV5 viremia and trunk shedding was documented in the other five elephants in the herd, who remained asymptomatic, except for 1 day of temporal gland swelling in an otherwise-healthy 1-yr-old calf. Unexpectedly, the two elephants most recently introduced into the herd 40 mo previously shed a distinctive EEHV5 strain from that seen in the other five elephants. This is the first report to document the kinetics of EEHV5 infection in captive Asian elephants and to provide evidence that this virus can cause illness in some animals.

Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression.

The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.

Modified vaccinia Ankara expressing EEHV1A glycoprotein B elicits humoral and cell-mediated immune responses in mice.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease (EEHV-HD) in Asian elephants and is the largest cause of death in captive juvenile Asian elephants in North America and Europe. EEHV-HD also has been documented in captive and wild elephants in their natural range countries. A safe and effective vaccine to prevent lethal EEHV infection would significantly improve conservation efforts for this endangered species. Recent studies from our laboratory suggest that EEHV morbidity and mortality are often associated with primary infection. Therefore, we aim to generate a vaccine, particularly for EEHV1 naïve animals, with the goal of preventing lethal EEHV-HD. To address this goal, we generated a Modified Vaccinia Ankara (MVA) recombinant virus expressing a truncated form of glycoprotein B (gBΔfur731) from EEHV1A, the strain associated with the majority of lethal EEHV cases. Vaccination of CD-1 mice with this recombinant virus induced robust antibody and polyfunctional T cell responses significantly above mice inoculated with wild-type MVA. Although the vaccine-induced T cell response was mainly observed in CD8+ T cell populations, the CD4+ T cell response was also polyfunctional. No adverse responses to vaccination were observed. Overall, our data demonstrates that MVA-gBΔfur731 stimulates robust humoral and cell-mediated responses, supporting its potential translation for use in elephants.

EEHV1A glycoprotein B subunit vaccine elicits humoral and cell-mediated immune responses in mice.

Asian elephants are an endangered species facing many threats, including severe hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV). EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North America and Europe, and also affects elephants in their natural range countries. Significant challenges exist for successful treatment of EEHV-HD, which include timely recognition of disease onset and limited availability of highly effective treatment options. To address this problem, our goal is to prevent lethal disease in young elephants by developing a vaccine that elicits robust and durable humoral and cell-mediated immunity against EEHV. EEHV glycoprotein B (gB) is a major target for cellular and humoral immunity in elephants previously exposed to EEHV. Therefore, we generated a vaccine containing recombinant EEHV1A gB together with a liposome formulated TLR-4 and saponin combination adjuvant (SLA-LSQ). CD-1 mice that received one or two vaccinations with the vaccine elicited significant anti-gB antibody and polyfunctional CD4+ and CD8+ T cell responses, while no adverse effects of vaccination were observed. Overall, our findings demonstrate that an adjuvanted gB protein subunit vaccine stimulates robust humoral and cell-mediated immune responses and supports its potential use in elephants.

Primary Infection May Be an Underlying Factor Contributing to Lethal Hemorrhagic Disease Caused by Elephant Endotheliotropic Herpesvirus 3 in African Elephants (Loxodonta africana).

Distinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection. IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations.

Nuclear-cytoplasmic shuttling is not required for the Epstein-Barr virus EBNA-LP transcriptional coactivation function.

Epstein-Barr virus (EBV) EBNA-LP is a transcriptional coactivator of EBNA2 that works though interaction with the promyelocytic leukemia nuclear-body-associated protein Sp100A. EBNA-LP localizes predominantly in the nucleus through the action of nuclear localization signals in the repeated regions of the protein. EBNA-LP has also been detected in the cytoplasm, and a previous study suggested that some of the EBNA-LP coactivation function is mediated by relocalizing histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. Although EBNA-LP can be found in the cytoplasm, it has no obvious nuclear export signal, and there is no direct evidence for active shuttling between these cellular compartments. Whether active shuttling between the nucleus and cytoplasm is required for coactivation remains to be clarified. To address these issues, we tested a variety of EBNA-LP isoforms and mutants for nuclear-cytoplasmic shuttling activity in an interspecies heterokaryon assay and for the ability to associate with HDAC4. EBNA-LP isoforms smaller than 42 kDa shuttle efficiently in the heterokaryon assay via a crm-1-independent mechanism. In addition, no specific EBNA-LP domain that mediates nuclear export could be identified. In contrast, an EBNA-LP 62-kDa isoform does not demonstrate detectable shuttling in the heterokaryon assay yet still coactivates EBNA2 similarly to the smaller EBNA-LP isoforms. All of the EBNA-LP mutants tested, including the coactivation-deficient DeltaCR3 mutant and the nonshuttling 62-kDa isoform, were capable of associating with HDAC4. Taken together, our results suggest that simple diffusion may account for the nuclear export observed with smaller isoforms of EBNA-LP, that nuclear-cytoplasmic shuttling is not required for efficient EBNA-LP coactivation function, and that competence for HDAC4 association is not sufficient to mediate nuclear-cytoplasmic shuttling or EBNA-LP coactivation in the absence of a functional interaction with Sp100A.