Extended genotypic evaluation and comparison of twenty-two cases of lethal EEHV1 hemorrhagic disease in wild and captive Asian elephants in India.

Thirteen new lethal cases of acute hemorrhagic disease (HD) with typical histopathogical features were identified in young Asian elephants (Elephas maximus indicus) in India between 2013 and 2017. Eight occurred amongst free-ranging wild herds, with three more in camp-raised orphans and two in captive-born calves. All were confirmed to have high levels of Elephant Endotheliotropic Herpesvirus type 1A (EEHV1A) DNA detected within gross pathological lesions from necropsy tissue by multi-locus PCR DNA sequencing. The strains involved were all significantly different from one another and from nine previously described cases from Southern India (which included one example of EEHV1B). Overall, eight selected dispersed PCR loci totaling up to 6.1-kb in size were analyzed for most of the 22 cases, with extensive subtype clustering data being obtained at four hypervariable gene loci. In addition to the previously identified U48(gH-TK) and U51(vGPCR1) gene loci, these included two newly identified E5(vGPCR5) and E54(vOX2-1) loci mapping far outside of the classic EEHV1A versus EEHV1B subtype chimeric domains and towards the novel end segments of the genome that had not been evaluated previously. The high levels of genetic divergence and mosaic scrambling observed between adjacent loci match closely to the overall range of divergence found within 45 analyzed North American and European cases, but include some common relatively unique polymorphic features and preferred subtypes that appear to distinguish most but not all Indian strains from both those in Thailand and those outside range countries. Furthermore, more than half of the Indian cases studied here involved calves living within wild herds, whereas nearly all other cases identified in Asia so far represent rescued camp orphans or captive-born calves.

Clinico-pathologic features of fatal disease attributed to new variants of endotheliotropic herpesviruses in two Asian elephants (Elephas maximus).

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

Modern evolutionary history of the human KSHV genome.

The genomes of several human herpesviruses, including Kaposi sarcoma (KS) herpesvirus (KSHV), display surprisingly high levels of both genetic diversity and clustered subtyping at certain loci. We have been interested in understanding this phenomenon with the hope that it might be a useful diagnostic tool for viral epidemiology, and that it might provide some insights about how these large viral genomes evolve over a relatively short timescale. To do so, we have carried out extensive PCR DNA sequence analysis across the genomes of 200 distinct KSHV samples collected from KS patients around the world. Here we review and summarize current understanding of the origins of KSHV variability, the spread of KSHV and its human hosts out of Africa, the existence of chimeric genomes, and the concept that different segments of the genome have had different evolutionary histories.

Comparison of genetic variability at multiple loci across the genomes of the major subtypes of Kaposi's sarcoma-associated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame K15 alleles at the right-hand end.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0. 35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized "orphan" region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.

High-level variability in the ORF-K1 membrane protein gene at the left end of the Kaposi's sarcoma-associated herpesvirus genome defines four major virus subtypes and multiple variants or clades in different human populations.

Infection with Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is common in certain parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere, except in AIDS patients. Nevertheless, HHV8 DNA is found consistently in nearly all classical, endemic, transplant and AIDS-associated KS lesions as well as in some rare AIDS-associated lymphomas. The concept that HHV8 genomes fall into several distinct subgroups has been confirmed and refined by PCR DNA sequence analysis of the ORF-K1 gene encoding a highly variable glycoprotein related to the immunoglobulin receptor family that maps at the extreme left-hand end of the HHV-8 genome. Among more than 60 different tumor samples from the United States, central Africa, Saudi Arabia, Taiwan, and New Zealand, amino acid substitutions were found at a total of 62% of the 289 amino acid positions. These variations defined four major subtypes and 13 distinct variants or clades similar to those found for the HIV ENV protein. The B and D subtype ORF-K1 proteins differ from the A and C subtypes by 30 and 24%, respectively, whereas A and C differ from each other by 15%. In all cases tested, multiple samples from the same patient were identical. Examples of the B subtype were found almost exclusively in KS patients from Africa or of African heritage, whereas the rare D subtypes were found only in KS patients of Pacific Island heritage. In contrast, C subtypes were found predominantly in classic KS and in iatrogenic and AIDS KS in the Middle East and Asia, whereas U.S. AIDS KS samples were primarily A1, A4, and C3 variants. We conclude that this unusually high diversity, in which 85% of the nucleotide changes lead to amino acid changes, reflects some unknown powerful biological selection process that has been acting preferentially on this early lytic cycle membrane signalling protein. Two distinct levels of ORF-K1 variability are recognizable. Subtype-specific variability indicative of long-term evolutionary divergence is both spread throughout the protein as well as concentrated within two 40-amino-acid extracellular domain variable regions (VR1 and VR2), whereas intratypic variability localizes predominantly within a single 25-amino-acid hypervariable Cys bridge loop and apparently represents much more recent changes that have occurred even within specific clades. In contrast, numerous extracellular domain glycosylation sites and Cys bridge residues as well as the ITAM motif in the cytoplasmic domain are fully conserved. Overall, we suggest that rather than being a newly acquired human pathogen, HHV8 is an ancient human virus that is preferentially transmitted in a familial fashion and is difficult to transmit horizontally in the absence of immunosuppression. The division into the four major HHV8 subgroups is probably the result of isolation and founder effects associated with the history of migration of modern human populations out of Africa over the past 35,000 to 60,000 years.

Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV8.

Strong serologic and molecular probe correlations indicate that the newly discovered gamma herpesvirus KSHV or HHV8 is the likely etiologic agent of all forms of Kaposi's sarcoma as well as BCBL/PEL and MCD in patients with acquired immunodeficiency syndrome (AIDS). Two large segments of HHV8 DNA from an AIDS-associated BCBL tumor covering genomic positions 0-52 kilobase [kb] and 108-140 kb have been cloned, mapped, and partially sequenced. Our studies have focused on novel viral proteins encoded within a 13-kb divergent locus (DL-B) by nine captured homologues of cellular genes, including vIL-6, vDHFR, vTS, vBcl-2, three C-C beta chemokines (vMIP-1A, vMIP-1B, and vBCK), and two LAP/PHD subclass zinc finger proteins (IE1A and IE1B). The HHV-8 vIL-6, vDHFR, vTS, and vBcl-2 proteins have all been shown to be active in a variety of appropriate functional assays, and transcripts from vIL-6, vMIP-1B, vIE1-A, vIE1-B, and vDHFR genes are all expressed as abundant single messenger RNA species after butyrate or phorbol ester (TPA) induction of the lytic cycle in HHV8-positive BCBL cell lines. All of these genes lie within a divergent transcriptional domain that contains a single central enhancer and associated untranslated leader region plus seven distinct proximal promoters, some of which are negatively regulated through AP-1 and ZRE motifs by the EBV ZTA transactivator. This region also encompasses a predicted complex oriLyt domain of 1050 bp that is duplicated in inverted orientation adjacent to the T0.7 latency RNA in another large divergent locus (DL-E). We have previously described three distinct subtypes of the HHV8 genome that differ by 1.0%-1.5% at the nucleotide level within the ORF26 and ORF75 genes. Certain strains or clades appear to have preferential geographic distributions, but it is not known as yet whether there are any specific disease associations. Interestingly, the A, B, and C subtypes of HHV-8 also proved to differ dramatically in coding content at both the extreme left and right ends of the unique segment of the genome as well as in the positions of the junctions with the terminal repeats. On the left-hand side, the receptor-like ORF-K1 protein is highly variable with A-strain subtypes displaying 15% amino acid differences from C strains and up to 30% differences from B strains. On the right-hand side, two unrelated alternative types of the putative multiple membrane spanning ORF-K15 protein are found.

Strain variability among Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genomes: evidence that a large cohort of United States AIDS patients may have been infected by a single common isolate.

Previous analysis of the majority of Kaposi's sarcoma (KS) tumors, in both AIDS and non-AIDS populations, has revealed the consistent presence of two small subsegments (open reading frame 25/26 [ORF25/26] and ORF75) of a novel human gamma class herpesvirus genome referred to as KSHV or HHV-8. We have carried out DNA sequence comparisons with DNAs encompassing a total of 2,500 bp each over three separate PCR-amplified fragments from KS lesions and body cavity-based lymphoma (BCBL) samples from 12 distinct patients, including four African and two classical or endemic non-AIDS KS samples. The results revealed differences at 37 of 2,500 nucleotide positions (i.e., 1.5% overall variation). However, the 12 HHV-8 genomes examined fell into three distinct but very narrow subgroupings (A, B, and C strains). All A strain isolates differed from B strain isolates at 16 positions, but of the eight U.S. samples tested, six were A strains, and these differed at no more than two positions among them. Similarly, three of the four African samples were B strains, which differed from each other at only one position. The two C strain genomes also displayed only one nucleotide variation, but they differed from all A strains at 26 positions and from all B strains at 20 positions. One C strain genome was present in all six independent lesions from an AIDS KS patient with disseminated disease, and the other represented a mosaic A/C recombinant genome from the HBL6 cell line derived from a BCBL tumor. Evaluation of previous data suggests that B and C strains may predominate in Africa and that A strains predominate in classical Mediterranean samples. Although both B and C strains are represented in U.S. AIDS patients, the majority (70 to 80%) of samples from the mid-East Coast region at least appear to be virtually identical, supporting the concept that they may all derive from the spread during the AIDS epidemic of a single recently transmitted infectious agent.

CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma and Hodgkin's disease.

The Epstein-Barr virus (EBV) latency C promoter drives expression of a family of viral proteins commonly targeted by CD8 cytotoxic T cells. These proteins are not generally expressed in African Burkitt's lymphoma and in EBV-associated Hodgkin's disease. The failure to express these proteins is almost certainly an important factor in the evasion of immunosurveillance by EBV-associated tumors. In a previous study, we have shown that transcriptional activation of the C promoter is inhibited by methylation of a particular CpG site upstream of the promoter that prevents binding of a cellular protein (CBF2), and we have shown that this and adjacent CpG sites are methylated in a Burkitt's lymphoma cell line. In the present study, we show that CpG sites in the CBF2 binding region are predominantly methylated in African Burkitt's lymphoma and in EBV-associated Hodgkin's disease. In addition, we present the first direct evidence that the C promoter is transcriptionally silent in Burkitt's lymphoma. In contrast, we show a complete absence of methylation in the CBF2 binding region in a case of reversible EBV-associated B-cell lymphoma arising in an immunocompromised patient whose tumor shows C promoter transcriptional activity. By inhibiting expression of highly antigenic viral proteins, methylation of transcriptional control sequences may veil the presence of virus in tumor tissue from CD8(+) cytotoxic T-cell immune surveillance and thus facilitate viral tumorigenesis.