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.

IDENTIFICATION OF TWO LETHAL CASES OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS HEMORRHAGIC DISEASE IN SUMATRAN ELEPHANT CALVES IN INDONESIA.

As many as a dozen cases of lethal acute hemorrhagic disease (HD) in young captive-born Sumatran sub-species Asian elephant (Elephas maximus sumatranus roman) calves raised naturally in camps in Sumatra have been observed in recent years. To address whether these deaths, like many others documented worldwide, might be associated with acute systemic infection by elephant endotheliotropic herpesvirus (EEHV), diagnostic polymerase chain reaction (PCR) tests followed by subtype deoxyribonucleic acid (DNA) sequencing analysis were carried out on pathologic tissue samples from two lethal HD cases that occurred within 6 days of one another in calves at the same camp. Viral DNA from five selected PCR loci was found to be present at high levels in both calves and proved to be the same EEHV1A virus species that has been described most commonly previously in numerous lethal or surviving symptomatic cases in North America, Europe, India, and Thailand. Furthermore, the two cases were identical at all five PCR loci tested (covering a total of 3,050 base pairs) and were therefore likely to have been infected from the same epidemiologic source herdmate. However, the strain involved (which was subtype-D2 in the vGPCR1 locus) differed from all previously characterized EEHV1A strains. In conclusion, these two calves are the first two confirmed HD cases in Sumatra alongside several other suspected HD cases in Sumatra that have succumbed to the same devastating EEHV1A-HD that has afflicted young Asian elephants worldwide over the past 25 yr. Because the progeny of some of the 1,500 remaining red-listed critically endangered Sumatra subspecies elephants are bred naturally in camps from wild parents it seems very likely that the EEHV1A herpesvirus identified here in these HD camp cases is also present in the free-ranging Sumatran elephant population, and this will have to be taken into account in future wildlife management policies and decisions.

Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease in Asian Elephant Calves in Logging Camps, Myanmar.

In recent years, an alarming number of cases of lethal acute hemorrhagic disease have occurred in Asian elephant calves raised in logging camps in Myanmar. To determine whether these deaths were associated with infection by elephant endotheliotropic herpesvirus (EEHV), we conducted diagnostic PCR subtype DNA sequencing analysis on necropsy tissue samples collected from 3 locations. We found that EEHV DNA from 7 PCR loci was present at high levels in all 3 calves and was the same EEHV1A virus type that has been described in North America, Europe, and other parts of Asia. However, when analyzed over 5,610 bp, the strains showed major differences from each other and from all previously characterized EEHV1A strains. We conclude that these 3 elephant calves in Myanmar died from the same herpesvirus disease that has afflicted young Asian elephants in other countries over the past 20 years.

The ubiquitin E3 ligase RAUL negatively regulates type i interferon through ubiquitination of the transcription factors IRF7 and IRF3.

In the course of combating infectious agents, type I interferon (IFN) needs a timely downregulation mechanism to avoid detrimental overreaction. Here we showed a mechanism for restraining type I IFN responses, which relied on a HECT domain ubiquitin (Ub) E3 ligase, RAUL. RAUL limited type I IFN production by directly catalyzing lysine 48-linked polyubiquitination of both interferon regulatory factor 7 (IRF7) and IRF3 followed by proteasome-dependent degradation. Suppression of RAUL by dominant-negative RAUL or siRNA augmented both basal and virus-induced production of type I IFN, which resulted in reduced viral replication. The Kaposi's sarcoma-associated herpes virus immediate-early lytic cycle trigger protein RTA recruited this mechanism to augment its countermeasures against the host antiviral response. These results unveil a previously unrecognized "brake mechanism" for type I IFN that maintains proper low amounts of type I IFN under physiological conditions and restrains its magnitude when the antiviral response intensifies.

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.

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

UNLABELLED: More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. IMPORTANCE: Acute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly involves a variety of strains of EEHV1, whose natural host has been unclear. Here, we carried out genotype analyses by partial PCR sequencing of necropsy tissue from five asymptomatic African elephants and identified multiple simultaneous infections by several different EEHV types, including high concentrations in lymphoid lung nodules. Overall, the results provide strong evidence that EEHV2, EEHV3, EEHV6, and EEHV7 represent natural ubiquitous infections in African elephants, whereas Asian elephants harbor EEHV1A, EEHV1B, EEHV4, and EEHV5. Although a single case of fatal cross-species infection by EEHV3 is known, the results do not support the previous concept that highly pathogenic EEHV1A crossed from African to Asian elephants in zoos.

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.

Rat cytomegalovirus (RCMV) English isolate and a newly identified Berlin isolate share similarities with but are separate as an anciently diverged clade from Mouse CMV and the Maastricht isolate of RCMV.

The genome of the rat cytomegalovirus (RCMV) English isolate (MuHV-8) differs significantly from the RCMV Maastricht isolate (MuHV-2) and other cytomegaloviruses (CMVs) in its size, base composition and genomic content. Analysis of the RCMV-Berlin isolate, MuHV-8, revealed that the two MuHV-8 isolates are highly similar in genome size and content, indicating that the smaller genome size (202 946 bp) compared to other known CMVs was not the result of an accidental deletion during passage in tissue culture. Surprisingly, the proteins encoded in MuHV-8 shared more overall similarity with their orthologues from mouse CMV (MuHV-1) compared to their orthologues in rat CMV (MuHV-2). Phylogenetic analyses of conserved viral genes showed that the two MuHV-8 isolates are from the same species and represent a unique clade that is distinct from other rodent CMVs.

Comparative genome analysis of four elephant endotheliotropic herpesviruses, EEHV3, EEHV4, EEHV5, and EEHV6, from cases of hemorrhagic disease or viremia.

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

Elephant endotheliotropic herpesviruses EEHV1A, EEHV1B, and EEHV2 from cases of hemorrhagic disease are highly diverged from other mammalian herpesviruses and may form a new subfamily.

UNLABELLED: A family of novel endotheliotropic herpesviruses (EEHVs) assigned to the genus Proboscivirus have been identified as the cause of fatal hemorrhagic disease in 70 young Asian elephants worldwide. Although EEHV cannot be grown in cell culture, we have determined a total of 378 kb of viral genomic DNA sequence directly from clinical tissue samples from six lethal cases and two survivors. Overall, the data obtained encompass 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus 10 novel genes, including a single large putative transcriptional regulatory protein (ORF-L). On the basis of differences in gene content and organization plus phylogenetic analyses of conserved core proteins that have just 20% to 50% or less identity to orthologues in other herpesviruses, we propose that EEHV1A, EEHV1B, and EEHV2 could be considered a new Deltaherpesvirinae subfamily of mammalian herpesviruses that evolved as an intermediate branch between the Betaherpesvirinae and Gammaherpesvirinae. Unlike cytomegaloviruses, EEHV genomes encode ribonucleotide kinase B subunit (RRB), thymidine kinase (TK), and UL9-like origin binding protein (OBP) proteins and have an alphaherpesvirus-like dyad symmetry Ori-Lyt domain. They also differ from all known betaherpesviruses by having a 40-kb large-scale inversion of core gene blocks I, II, and III. EEHV1 and EEHV2 DNA differ uniformly by more than 25%, but EEHV1 clusters into two major subgroups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three nonadjacent loci totaling 15 kb diverge by between 21 and 37%. One strain of EEHV1B analyzed is interpreted to be a modern partial recombinant with EEHV1A. IMPORTANCE: Asian elephants are an endangered species whose survival is under extreme pressure in wild range countries and whose captive breeding populations in zoos are not self-sustaining. In 1999, a novel class of herpesviruses called EEHVs was discovered. These viruses have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the United States and Europe since 1980. The disease is increasingly being recognized in Asian range countries as well. These viruses cannot be grown in cell culture, but by direct PCR DNA sequence analysis from segments totaling 15 to 30% of the genomes from blood or necropsy tissue from eight different cases, we have determined that they fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, and we propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

Complete genome sequence of the english isolate of rat cytomegalovirus (Murid herpesvirus 8).

The complete genome of the English isolate of rat cytomegalovirus (RCMV-E) was determined. RCMV-E has a 202,946-bp genome with noninverting repeats but without terminal repeats. Thus, it differs significantly in size and genomic arrangement from closely related rodent cytomegaloviruses (CMVs). To account for the differences between the rat CMV isolates of Maastricht and England, RCMV-E was classified as Murid herpesvirus 8 by the International Committee on Taxonomy of Viruses.

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.

KSHV regulation of fibulin-2 in Kaposi's sarcoma: implications for tumorigenesis.

Kaposi's sarcoma is an angioproliferative tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection of vascular endothelial cells. Fibulins, proteins that associate with extracellular matrix (ECM) proteins, may have both tumor-suppressive and oncogenic activities. We found that the expression of fibulin-2 protein and mRNA were decreased 50-fold and 26-fold, respectively, in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC). Using quantitative RT-PCR, we found a fivefold and 25-fold decrease of fibulin-2 extracellular matrix binding partners, fibronectin and tropoelastin, respectively. Time-course transcriptional analyses over 10 days showed that in addition to that of fibulin-2, expression of fibulins 3 and 5 was decreased in KSHV-infected DMVEC, fibulins 1C/1D were increased, and fibulins 4, 6, and 7 were unchanged. KSHV latency-associated nuclear antigen (LANA) transcription levels rose consistently over the same period. Addition of recombinant fibulin-3 or -5 for 48 hours to 10-day KSHV-infected cells caused a suppression of KSHV-induced vascular endothelial growth factor (VEGF) protein and mRNA levels. Recombinant fibulin-3 also significantly reduced VEGF receptor 3 expression. In pleural effusion lymphoma cell lines that express variable levels of KSHV lytic replication, we observed no detectable fibulin-2 or -5 expression. Finally, fibulin-2 expression was decreased in tissue microarrays from KSHV-infected, LANA-positive patient cells as compared to that in patient nontumor controls. Understanding the interactions between KSHV and the fibulins may lead to the development of novel therapies for treatment of Kaposi's sarcoma.

Initiation of angiogenic Kaposi's sarcoma lesions.

A new mouse model reported in this issue of Cancer Cell implies that VEGF/KDR-mediated paracrine effects induced by the lytic cycle vGPCR signaling protein encoded by human Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) may be involved in promoting the proliferation of the KSHV latently infected spindle endothelial cells of Kaposi's sarcoma.

Comparison of humoral immune responses to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus using a viral proteome microarray.

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). METHODS: To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi's sarcoma or lymphoma. RESULTS: In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1-positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. CONCLUSIONS: The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

Recombinant luciferase-expressing human cytomegalovirus (CMV) for evaluation of CMV inhibitors.

Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.

A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency.

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.

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.

KSHV downregulation of galectin-3 in Kaposi's sarcoma.

Galectins are a family of proteins that share an affinity for beta-galactoside containing glycoconjugates. In prostate, ovarian and breast cancer, downregulation of galectin-3 is associated with malignancy and tumor progression. Kaposi's sarcoma (KS) is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of primary effusion lymphomas and some forms of multicentric Castleman's disease. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS. We found reduced levels of galectin-3 expression in a significant fraction of latency-associated nuclear antigen (LANA)-positive spindle cell regions in human archival KS tissue and as measured in KS tissue microarrays. Here we demonstrate that galectin-3 protein expression is downregulated 10-fold in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC) accompanied by downregulation of message. There is loss of galectin-3 staining in KSHV-infected DMVEC by dual labeled immunohistochemistry in LANA-positive spindle cells. We observed a consistent downregulation of galectin-3 by time-course transcriptional analysis. Of the galectins assayed, only galectin-1 was also downregulated in KSHV-infected DMVEC. We examined 86 KS tumors; 19 were LANA positive (22%) and 67 LANA negative (78%). All 86 tumors were found to be galectin-3 positive; 11 of 19 showed reduced expression of galectin-3 in LANA-positive spindle cell regions. Our data suggest that KSHV vFLIP and LANA are the viral genes targeting galectin-3 downregulation. The contribution of host factors to the pathogenesis of KS is essential for early detection and development of innovative therapies for treatment.