Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.

Human herpesvirus-8 (HHV-8) has been detected in Kaposi's sarcoma (KS) lesions of all types (AIDS-related, classical and endemic), in body-cavity-based B-cell lymphomas (BCBLs) and in lesions of multicentric Castleman's disease (MCD). We have identified a major gamma-herpesvirus-divergent locus (DL-B) in HHV-8 DNA encoding several HHV-8 unique open reading frames (ORFs), including a homologue of interleukin-6 (IL-6) and two homologues of macrophage inflammatory protein MIP-1. We show that the HHV-8-encoded IL-6 homologue (vIL-6) shares functional properties with endogenous IL-6 proteins and that both vIL-6 and vMIP-1 transcripts are present at high levels following butyrate induction of an HHV-8' BCBL cell line. Low amounts of constitutive vIL-6, but not vMIP-1, mRNA were also detected. The presence of a functional IL-6 homologue encoded by HHV-8 may provide a mechanistic model for the hypothesized role of HHV-8 in KS, MCD and BCBL that involves the mitogenic effects of vIL-6 on surrounding cells. MIP-1 proteins may enhance these effects through the chemotactic recruitment of endogenous cytokine-producing cells into affected tissues and could potentially influence HIV disease progression in coinfected individuals through interactions with the HIV co-receptor CCR-5.

Novel endotheliotropic herpesviruses fatal for Asian and African elephants.

A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease.

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.

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.

A cytomegalovirus DNA sequence containing tracts of tandemly repeated CA dinucleotides hybridizes to highly repetitive dispersed elements in mammalian cell genomes.

A single 880-base-pair region within the genome of simian cytomegalovirus strain Colburn contains sequences that hybridize intensely with both human and mouse total genome DNA probes. This sequence was also found in a second simian cytomegalovirus isolate and was retained in both plaque-purified virus subclones and in plasmid DNA clones containing the SalI P fragment. Cleaved genomic DNAs from several mammalian species all exhibited strong dispersed hybridization with the SalI-P probes, and over 70% of the lambda clones in a mouse genomic library plus several selected clones containing globin, 45S rDNA, or 5S rDNA genes all formed hybrids with SalI-P. The appropriate region of cytomegalovirus SalI-P contains relatively A + T-rich unique sequences interrupted by three stretches of the simple alternating dinucleotides, (CA)15, (CA)22, and (CA)21, which we show to be responsible for most of the cell-virus homology. We conclude that discrete, tandemly repeated (CA) dinucleotide tracts capable of forming left-handed Z-DNA helices punctuate mammalian genomes at greater than 10(5) copies per cell and that three adjacent copies of what appear to be a family of interspersed repetitive elements containing these (CA)n stretches are carried in the genomes of simian cytomegaloviruses.

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING finger-dependent cryptic transactivator function of PML.

Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.

In situ detection of human cytomegalovirus immediate-early gene transcripts within cardiac myocytes of patients with HIV-associated cardiomyopathy.

OBJECTIVE: Recent clinical and echocardiographic studies have identified dilated cardiomyopathy in 10-20% of HIV-infected adults. The purpose of this study was to determine the role of cardiotropic cytomegalovirus (CMV) infection in the development of HIV-associated cardiomyopathy. DESIGN: We generated sense and antisense digoxigenin-labeled riboprobes derived from the CMV immediate-early (IE) and delayed-early (DE) genes and applied them retrospectively to endomyocardial biopsy samples and control autopsy cardiac samples from HIV-infected patients. SETTING: Tertiary care, referral hospital. PATIENTS: Twelve consecutive HIV-infected patients with global left ventricular hypokinesis demonstrated on two-dimensional echocardiography; eight randomly selected control autopsy cardiac samples from HIV-infected patients without cardiac disease during life. MEASUREMENTS AND MAIN RESULTS: Of the 12 endomyocardial biopsy specimens, six (50%) were found to have specific myocyte nuclear and perinuclear hybridization for transcripts of the CMV IE gene, consistent with non-permissive or latent infection. Similar patterns were not found in any of the eight autopsy control samples. All six patients presented with unexplained congestive heart failure and had CD4 counts less than 100 x 10(6)/l; all six biopsy samples had immunohistochemical evidence of increased myocardial major histocompatibility complex (MHC) class I expression, a finding typical of non-HIV myocarditis. None of the endomyocardial biopsy samples had characteristic CMV inclusions and no specific hybridization was noted with the DE gene riboprobe, suggesting that no active viral DNA replication was present. Only two of the six patients with myocyte hybridization with the IE riboprobe had clinical evidence of solid organ infection with CMV at the time of cardiovascular presentation. CONCLUSIONS: This study is the first to demonstrate the expression of the IE gene of CMV within myocytes from HIV-infected patients with cardiomyopathy, suggesting a non-permissive infection of myocytes without classical intranuclear inclusions. Myocyte infection may be necessary to trigger cellular and humoral-mediated cardiac injury and may be best identified using in situ hybridization techniques.

Structural organization of human herpesvirus DNA molecules.

The herpesviruses are among the largest and most complex of all DNA viruses, and their genomes display an astonishing diversity in size, structure, and organization. In 1974, the features of large inverted repeats and structural isomerization were first discovered, and these proved to be characteristic properties of many herpesvirus genomes. Since then, research using the powerful techniques of modern molecular biology has revealed a great deal of comparative structural information about the arrangement of repetitive sequences and the location, structure, and primary nucleotide sequences of the genes for several easily assayed or abundantly expressed gene products. Extensive restriction enzyme cleavage maps and complete sets of cloned DNA fragments have been constructed for each of the five human herpesviruses, HSV-1, HSV-2, CMV, EBV, and VZV, and the entire 175,000-bp nucleotide sequence of EBV DNA has been determined. Based on these maps and reagents, the procedures of "DNA fingerprinting" and "dot hybridization" are proving useful at a clinical level for characterization of isolates and studying herpesvirus epidemiology. Strain differences, localized heterogeneity, tandem-repeat-defective genomes, and sites of cell-virus DNA homology have been described in some detail. The attention of basic researchers is now turning to equating structure with function, and rapid progress is expected in studies aimed at a better understanding of the mechanisms of viral DNA replication, maintenance of the latent state, reactivation, transformation, packaging, and regulation of the lytic cycle, etc using cloned functionally active DNA fragments, isolated intact genes and promoters, and DNA transfection and in vitro expression systems.

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.

Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays.

The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in-vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro. Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells.

The WAF1-mediated p53 growth-suppressor pathway is intact in the coronary arteries of heart transplant recipients.

It has been suggested that the interaction of cytomegalovirus (CMV) with the p53 tumor suppressor gene product plays a role in the development of coronary artery restenosis after angioplasty. CMV nucleic acids have been observed in the coronary arteries of allografted hearts, suggesting a possible role for the interaction of CMV with p53 in the development of accelerated graft arteriosclerosis in transplant recipients. Formalin-fixed, paraffin-embedded sections of coronary arteries from 19 transplanted hearts were immunostained for the p53 gene product using Target Unmasking Fluid (TUF)-mediated immunohistochemistry and the anti-p53 antibodies CM1 and DO7. Fresh-frozen sections of coronary arteries were also available from six of the 19 hearts, and these fresh-frozen sections were immunostained for the p53 gene product with the DO7 antibody and for WAF1 using the anti-WAF1 antibody EA10. Focal and weak staining for p53 was observed in smooth muscle and endothelial cells in two of 19 vessels, whereas the remaining 17 did not stain. CMV nucleic acids were previously shown in six of 13 of these hearts by in situ hybridization. The fresh-frozen sections of coronary arteries also did not stain for p53, but the smooth muscle cells in these vessels did stain intensely for WAF1. These results suggest three possibilities: (1) CMV-p53 interactions are not important in the development of accelerated graft arteriosclerosis; or (2) there is an interaction, but it is transient and not detectable at the time points examined in this study; or (3) there is an interaction, but binding of CMV to p53 leads to accelerated degradation of p53, as occurs with HPV-E6. The expression of WAF1 further suggests that the WAF1-mediated antiproliferative signal is intact in these vessels.

A survey of Epstein-Barr virus DNA in lymphoid tissue. Frequent detection in Hodgkin's disease.

A total of 151 unselected malignant and nonmalignant lymphoid tissue samples were surveyed by Southern blotting for the presence of Epstein-Barr virus (EBV) DNA. Eight of 28 Hodgkin's disease (HD) samples (29%) had detectable EBV DNA. Both nodular sclerosis and mixed cellularity histologic results were positive. The tumor type with the next highest frequency, 8%, was diffuse large cell lymphoma. The presence of EBV DNA in some HD biopsies suggests that EBV may be a factor in the pathogenesis of this disease. Alternatively, its presence may be secondary to the immune deficiency characteristic of HD. The clonal B-lymphocyte expansions reported in some cases of HD may result from EBV infection.

Multiple Tandemly Repeated Binding Sites for the YY1 Repressor and Transcription Factors AP-1 and SP-1 Are Clustered within Intron-1 of the Gene Encoding the IE110 Transactivator of Herpes simplex Virus Type 1.

Expression of the IE110 (ICP0) transactivator protein of HSV appears to be critical for reactivation from the latent state and occurs at immediate-early times during the lytic cycle under the control of an upstream divergent enhancer-promoter region that contains multiple Oct and Sp-1 binding sites overlapping with VP16 response elements. Surprisingly, the large 800-bp first intron of the HSV-1 IE110 gene also proved to have a complex repetitive organization encompassing multiple transcription factor binding sites within four distinct domains. DNaseI footprinting studies revealed that 13 of 17 copies of a 15-bp repeated element represented high-affinity binding sites for the cellular YY1 repressor protein. Between 4 and 7 of these sites are direct tandem repeats and the rest are interpersed with three repeated AT-rich motifs and a dyad symmetry region containing two strong AP-1 binding sites and an adjacent SP-1 binding site on each arm. Several of the YY1 sites also bound weakly to SRF. The intron also contains four clustered purine/pyrimidine tracts of between 16 and 23 bp long. Both the AP-1/AP-2/SP-1 dyad protein binding region and, to a lesser extent, the YY1 tandem-repeat cluster conferred responsiveness to TPA when placed upstream of a heterologous promoter in transient expression assays. The functional significance of the HSV-1 IE110 intron region is unknown as yet, but the novel arrangement of tandemly repeated YY1 sites has the potential to produce structural bending and transcriptional attenuation effects. Interestingly, few of these transcription factor binding motifs are conserved in the equivalent IE110 intron of HSV-2, and the domain appears to represent a unique alternative control region that is specific for HSV-1. Copyright 1995 S. Karger AG, Basel

Structural Organization of the Spliced Immediate-Early Gene Complex that Encodes the Major Acidic Nuclear (IE1) and Transactivator (IE2) Proteins of African Green Monkey Cytomegalovirus.

Total immediate-early (IE) RNA synthesized after infection with African green monkey cytomegalovirus (SCMV) in the presence of cycloheximide contained a major 2.3-kb mRNA species that acted as template for in vitro synthesis of a single 94-kD nuclear protein. The same IE RNA hybridized predominantly to a 1.8-kb subregion of the 220-kb genome which mapped 1.5 kb to the left of the in vitro transcription start site and TATATAA motif previously associated with the powerful MIE (IE94) enhancer region. However, DNA sequence and S1-mapping analysis of a 5-kb region downstream from the promoter revealed the existence of a far upstream noncoding first exon and four additional spliced exons capable of encoding two alternative protein products with shared N-terminal domains. This region is similar in structure to that of the MIE gene complex of human cytomegalovirus (HCMV), including being highly CpG suppressed. Exons 2, 3, and 4 encode an acidic protein equivalent to the 68-kD IE1 protein (UL123) of HCMV and exons 2, 3, and 5 encode a protein equivalent to the 80-kD IE2 (UL122) DNA-binding protein of HCMV. Transcripts from across the IE2 region were detected within the cycloheximide RNA, but they were present at 10- to 20-fold lower abundance than IE1 transcripts. The proposed 547-codon IE1 (IE94) acidic phosphoprotein of SCMV displays minimal residual homology with the IE1 protein of HCMV, but both associate with metaphase chromosomes and have large C-terminal glutamic-acid-rich domains. In contrast, the proposed 583-codon IE2 protein of SCMV displays extensive amino acid similarity to the HCMV IE2 transcriptional regulatory protein especially within C-terminal domains that are known to play a major role in promoter targeting for both transactivation and negative autoregulation functions. Copyright 1995 S. Karger AG, Basel

Detection of the Human Cytomegalovirus 2.0-kb Immediate Early Gene 1 Transcripts in Permissive and Nonpermissive Infections by RNA in situ Hybridization.

The immediate early gene 1 (IE1) is the first gene to be expressed following the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. Therefore, the IE1 gene is a potential candidate for the development of probes to detect HCMV in various states of infection. Using strand-specific (32)P- or digoxigenin-labeled riboprobes derived from an exon-specific subgenomic fragment of the HCMV Towne IE1 gene, we performed Northern blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By Northern blot analysis and by in situ hybridization, expression of the 2.0-kb IE1 gene was found in permissive as well as in nonpermissive infections. Specific nuclear and cytoplasmic hybridization was found at 5, 10, 24 and 72 h after infection in human fibroblasts. In comparison, hybridization was first detected at 10 h after infection in mouse fibroblasts. Hybridization with the IE1 probe was detected in cells with and without cytopathic changes in the formalin-fixed paraffin-embedded HCMV-infected human tissues. Hybridization patterns of the IE1 riboprobe were compared to those of the HCMV 2.7-kb major early beta-riboprobe which we have previously described [Am J Pathol 141:1247-1254;1992]. Although both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results suggest that RNA in situ hybridization with a probe directed at the IE1 transcripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections. Copyright 1997 S. Karger AG, Basel

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines.

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.

A molecular hybridization study for the presence of Herpes simplex, cytomegalovirus and Epstein-Barr virus in brain and blood of Alzheimer's disease patients.

Among several hypothesis for the development of Alzheimer's disease is a viral hypothesis. The present study was designed to detect nucleic acid sequences for conventional viruses in peripheral blood cells and brain of Alzheimer's disease patients. DNA was isolated from peripheral blood cells and brain tissue from control individuals and Alzheimer's disease patients. Southern blot analysis was performed using radiolabeled probes for various conventional viruses. The results fail to detect the presence of Herpes simplex 1 (HSV-I), Herpes simplex II (HSV-II), Epstein-Barr virus (EBV) and cytomegalo virus (CMV) at a sensitivity level of detecting 1 genome/100 cells. We exclude conventional viruses as a cause of Alzheimer's disease at this level of detection.

Clinical and pathological findings of a newly recognized disease of elephants caused by endotheliotropic herpesviruses.

The unique clinical and pathological findings in nine Asian (Elephas maximus) and two African (Loxodonta africana) elephants from North American Zoos with a highly fatal disease caused by novel endotheliotropic herpesviruses are described. Identification of the viruses by molecular techniques and some epidemiological aspects of the disease were previously reported. Consensus primer polymerase chain reaction (PCR) combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and the second in African elephants. Disease onset was acute, with lethargy, edema of the head and thoracic limbs, oral ulceration and cyanosis of the tongue followed by death of most animals in 1 to 7 days. Pertinent laboratory findings in two of three clinically evaluated animals included lymphocytopenia and thrombocytopenia. Two affected young Asian elephants recovered after a 3 to 4 wk course of therapy with the anti-herpesvirus drug famciclovir. Necropsy findings in the fatal cases included pericardial effusion and extensive petechial hemorrhages in the heart and throughout the peritoneal cavity, hepatomegaly, cyanosis of the tongue, intestinal hemorrhage, and ulceration. Histologically, there were extensive microhemorrhages and edema throughout the myocardium and mild, subacute myocarditis. Similar hemorrhagic lesions with inflammation were evident in the tongue, liver, and large intestine. Lesions in these target organs were accompanied by amphophilic to basophilic intranuclear viral inclusion bodies in capillary endothelial cells. Transmission electron microscopy of the endothelial inclusion bodies revealed 80 to 92 nm diameter viral capsids consistent with herpesvirus morphology. The short course of the herpesvirus infections, with sudden deaths in all but the two surviving elephants, was ascribed to acute cardiac failure attributed to herpesvirus-induced capillary injury with extensive myocardial hemorrhage and edema.