Delta Ag Research Articles

Prevalence of Hepatitis Delta Virus (HDV) Infection in Chronic Hepatitis B Patients with Unusual Clinical Pictures

BackgroundProbably 5% of the HBV carriers have HDV super infection. The risk of fulminant hepatitis, cirrhosis and hepatocellular carcinoma is higher in superinfection than the settings when HBV is alone.ObjectivesThe aim of this study was to evaluate the prevalence of HDV in Iranian HBV isolates and to compare their clinical and virological pictures as well as their HDV genetic variations with other worldwide isolates.Patients and Methods81 carriers with positive results for HBsAg with upper limit ranges of ALT and low or undetectable levels of HBV viral load who did not respond to HBV therapy were selected. After RT amplification of HDV Delta antigen, direct sequencing and phylogenetic study were performed to explore the genotype(s) and nucleotide/amino acid variations.Results12 (14.8%) patients had positive results for both HDV RNA and anti-HDV. The mean ALT level was higher in HDV positive patients (75.9 U/ML) than HBV-mono-infected individuals; however, the mean HBV viral load was lower in coinfected patients than HBV-mono-infected patients. Phylogenetically, genotype I was the only detected genotype, and the most closely related isolates were of Turkish, Italian and Mongolian origin. Within the delta Ag, there were 326 nucleotide mutations, of which 111 and 215 were silent and missense, respectively. The total number of amino acid substitution was 148; most were located in known functional/epitopic domains. There was no correlation between the numbers of amino acid mutations, with clinical, virological status of the patients.ConclusionsHDV should be suspected in HBV carriers with unusual clinical and virological pictures. Relatedness of Iranian HDV isolates to Italian and Turkish sequences proposed a common Caucasian origin for the distribution of HDV genotype I in this ethnic group.

Six novel mutations in the emerin gene causing X-linked Emery-Dreifuss muscular dystrophy.

Mutations in the emerin gene, also referred to as the STA- or EMD-gene, have been found to be the cause of X-linked Emery-Dreifuss muscular dystrophy (EMD). For the present study an optimized set of primers was designed to amplify and sequence each of the six emerin gene exons, including the intron/exon boundaries. All emerin gene exons of 30 unrelated EMD patients have been screened by heteroduplex analysis. Aberrant patterns of single exons were found in seven patients. Direct sequencing of the respective exons revealed six novel mutations distributed in the promotor region and exons 3-6 (delta nt -19 to -40; delta AG nt 620-621; ins A nt 895; delta AT nt 908-909; C-->A nt 1420; ins TA nt 1570). By this study, the first mutations in the promotor region and in exon 5 have been identified. Each of the 25 mutations that have been described so far, including those from the present study, abolishes the synthesis of functional emerin. The mutations were submitted to the EMD Mutation database (http://www.path.cam.ac.uk/emd).

Six novel mutations in the emerin gene causing X‐linked Emery‐Dreifuss muscular dystrophy

Mutations in the emerin gene, also referred to as the STA- or EMD-gene, have been found to be the cause of X-linked Emery-Dreifuss muscular dystrophy (EMD). For the present study an optimized set of primers was designed to amplify and sequence each of the six emerin gene exons, including the intron/exon boundaries. All emerin gene exons of 30 unrelated EMD patients have been screened by heteroduplex analysis. Aberrant patterns of single exons were found in seven patients. Direct sequencing of the respective exons revealed six novel mutations distributed in the promotor region and exons 3-6 (delta nt -19 to -40; delta AG nt 620-621; ins A nt 895; delta AT nt 908-909; C-->A nt 1420; ins TA nt 1570). By this study, the first mutations in the promotor region and in exon 5 have been identified. Each of the 25 mutations that have been described so far, including those from the present study, abolishes the synthesis of functional emerin. The mutations were submitted to the EMD Mutation database (http://www.path.cam.ac.uk/emd).

Epitopes exposed on hepatitis delta virus ribonucleoproteins

A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.

Hepatitis delta virus mutant: effect on RNA editing.

During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.

Gamma delta T cells can recognize nonclassical MHC in the absence of conventional antigenic peptides.

Although T cells expressing the gamma delta Ag receptor (gamma delta cells) have been found in all organisms with lymphoid systems, little is understood about their immunologic function. It is known that T cells that express the alpha beta Ag receptor (alpha beta cells) fight infection by recognizing foreign Ag as peptide presented by MHC molecules; however, it is not known how gamma delta cells recognize Ag. Characterization of ligands of the gamma delta cell Ag receptor (TCR) could lead to a better understanding of gamma delta cell function. We have characterized the ligand of the G8 gamma delta cell; we find that G8 gamma delta cells are stimulated by T22, a nonclassical class I MHC molecule. To study the involvement of peptides in gamma delta cell recognition, T22 was expressed in cells deficient in peptide transporter function and in Drosophila cells, which can express MHC molecules without associated peptides. We find no evidence that G8 gamma delta cells recognize peptides bound to T22. These results suggest that some gamma delta cells, unlike alpha beta cells, may not be restricted to recognition of peptide-MHC complexes.

Hepatitis delta virus replication in vitro is not affected by interferon- or - despite intact cellular responses to interferon and dsRNA

The hepatitis delta virus (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like structure. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with anti-viral activity, namely the dsRNA-dependent protein kinase (PKR) and 2',5' oligoadenylate (2',5' A) synthetase. Since the virus replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the virus may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA construct, with IFN-alpha or IFN-gamma for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2',5' A synthetase and class I MHC is normal and cotransfection of a construct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase construct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic viruses is unaffected. IFN-beta is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-alpha or IFN-gamma, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.

Diagnosis of Mixed Acid-Base Disordersin Diabetic Ketoacidosis

In diabetic ketoacidosis, a mixed acid-base disorder is suggested when the anion gap increase (delta AG) does not equal the bicarbonate decrease (delta HCO3), or when the delta AG/delta HCO3 ratio does not equal 1.0. It is widely assumed that delta AG/delta HCO3 is significantly different from 1.0 when it is less than 0.8 or greater than 1.2. The validity of these ratio limits were examined by analyzing a normal control group of 68 subjects and 27 diabetic ketoacidosis admissions that had no evidence of mixed disorders. In the 27 ketoacidosis admissions, regression analysis showed that delta AG was predicted to equal delta HCO3, as expected in pure anion gap acidosis: delta AG = 1.0 delta HCO3 (r = 0.744, p < 0.001). It was found that delta AG is significantly different from delta HCO3 when they differ by more than 8 mEq/L, and equivalently, delta AG/delta HCO3 is significantly different from 1.0 when it is less than (1.0 - 8/delta HCO3) or greater than (1.0 + 8/delta HCO3). These criteria from regression analysis suggested that 4% of the 27 pure anion gap acidoses, and 3% of the control group, had mixed disorders. In contrast, the ratio limits of 0.8 and 1.2 suggested 56% of the pure anion gap acidoses, and 94% of the control group, had mixed disorders. It was concluded that mixed disorders are overdiagnosed by the ratio limits of 0.8 and 1.2. Mixed disorders are more accurately detected by noting whether delta AG and delta HCO3 differ by more than 8 mEq/L.

The role of viral enhancer "core" motif-related sequences in regulating T cell receptor-gamma and -delta gene expression.

T cells express clonally distributed alpha beta or gamma delta Ag receptor heterodimers. Transcriptional enhancers for the genes of all four subunits are active in both gamma delta and alpha beta T cells, but are less active or inactive in other cells. Conserved sequence motifs are present in all four enhancers, suggesting that common transcription factors regulate TCR gene expression. One of these motifs in the gamma 3 site of the TCR-gamma enhancer is similar to motifs found in several other lymphoid-specific and viral enhancers. This conserved "core" sequence is present in the enhancers of Moloney and SL3-3 murine leukemia viruses, important for transcription in T cells and in determining disease specificity. Here we characterize the gamma 3 site of the gamma enhancer and a corresponding homologous site, delta E3, of the TCR-delta enhancer. Our results suggest that the core site is critical for activity of the 200-bp gamma enhancer fragment and of the gamma 3 and delta E3 sites. Furthermore, we identify a nuclear factor in human T cell lines that specifically binds the core region in these and several other core-containing enhancers. This factor may be identical to or related to a purified bovine nuclear core binding factor that binds the core region of the Moloney murine leukemia virus enhancer, gamma 3 and delta E3 sites, suggesting that similar proteins regulate the TCR-gamma, delta and Moloney murine leukemia virus enhancers. Other sequences in the gamma 3 site upstream of the core sequence are also critical for activity in T cells, suggesting that at least two different factors are required for functional activity of the gamma 3 site.

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.

Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 δ and ε but not membrane CD3

Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3- CD16- cells were sorted and cloned by limiting dilution. Two representative clones, FL121 and FL125, were expanded and characterized. They shared the phenotype of CD2+CD3-CD4-CD5-CD6+CD16-CD56+. FL121 did not express CD8 whereas FL125 expressed CD8 alpha but not CD8 beta. Both clones were found to express cytoplasmic CD3 delta and CD3 epsilon Ag while CD3- NK clones isolated from PBL were negative for them. These results indicate that FL121 and FL125 were committed to the T-cell lineage. Southern blot analysis showed that the TCR beta genes and the TCR gamma genes of these clones were in the germ-line configuration. The establishment of FL121 and FL125 may provide a novel insight into the earliest stage of T-cell development in man.

Hepatitis delta antigen: antigenic structure and humoral immune response

Hepatitis delta virus (HDV) is a small RNA virus that is dependent on helper functions provided by hepatitis B virus. The hepatitis delta Ag (HDAg) is the only protein known to be made from the viral genome, from an ORF with a coding capacity of 214 amino acids. The immunogenic epitopes of HDAg and the immune response to it were mapped by the use of synthetic peptides, antipeptide antibodies, and human mAb. Antipeptide sera covering approximately 60% of the linear sequence reacted with liver-derived HDAg. Antisera from HDV-infected humans, chimpanzees, and woodchucks reacted with from 2 to 13 of 15 peptides. The epitopes of two human anti-HD mAb were mapped to overlapping but distinct epitopes in the region around residues 106-123. Sera from infected humans, chimpanzees, and woodchucks were also tested by competition with the mAb. Use of the peptides and antipeptide sera defined one region in the sequence (residues 52-93) which is immunodominant in the immune response to HDAg. Reactivity of both peptides and antipeptide antibodies was very broad, covering most or all of the linear sequence. Competition assays also provided information on conformational epitopes, as well as the sequential epitopes defined by direct assays. The peptides and antipeptide antibodies should be useful in new assay development, in dissecting the anti-HD response in terms of chronic vs self-limited infection, and in studying the role of anti-HD in infection and recovery.

Antigenic markers of hepatocellular carcinoma

One hundred thirty-five hepatocellular carcinomas were examined for the presence of antigenic tumor markers by the avidin-biotin-peroxidase complex method. Ninety-seven were from the US and 38 came from Argentina. The following markers were tested: alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis D delta antigen (HD delta Ag), and Mallory's bodies (MB). In the US cases, AFP was present in 43%, AAT in 41%, HBsAg in 17%, and MB in 48%. Both HBcAg and HD delta Ag were absent. In the cases from Argentina, AFP was found in 26% and AAT in 18%. None of the other antigens were seen. Thirteen US tumors expressed three antigens and two four antigens simultaneously. This study reveals in humans a heterogenous expression of antigens by neoplastic hepatocytes with geographic differences, possibly due to multiple factors such as alcohol consumption or prevalence of hepatitis B infection.

Hepatitis delta antigen. Antigenic structure and humoral immune response.

Hepatitis delta virus (HDV) is a small RNA virus that is dependent on helper functions provided by hepatitis B virus. The hepatitis delta Ag (HDAg) is the only protein known to be made from the viral genome, from an ORF with a coding capacity of 214 amino acids. The immunogenic epitopes of HDAg and the immune response to it were mapped by the use of synthetic peptides, antipeptide antibodies, and human mAb. Antipeptide sera covering approximately 60% of the linear sequence reacted with liver-derived HDAg. Antisera from HDV-infected humans, chimpanzees, and woodchucks reacted with from 2 to 13 of 15 peptides. The epitopes of two human anti-HD mAb were mapped to overlapping but distinct epitopes in the region around residues 106-123. Sera from infected humans, chimpanzees, and woodchucks were also tested by competition with the mAb. Use of the peptides and antipeptide sera defined one region in the sequence (residues 52-93) which is immunodominant in the immune response to HDAg. Reactivity of both peptides and antipeptide antibodies was very broad, covering most or all of the linear sequence. Competition assays also provided information on conformational epitopes, as well as the sequential epitopes defined by direct assays. The peptides and antipeptide antibodies should be useful in new assay development, in dissecting the anti-HD response in terms of chronic vs self-limited infection, and in studying the role of anti-HD in infection and recovery.

Liver histopathology in autopsied drug-addicts

Liver specimens of 150 randomly selected autopsied drug addicts from the province of Milan (Italy) were studied using light microscopy, polarized light and immunohistochemical methods. Simple histological changes and the main diagnoses are described. The main histological diagnoses in order of frequency were: non-specific reactive hepatitis (NSRH) (52%), chronic aggressive hepatitis (CAH) (24%), acute hepatitis (AH) (12%), normal liver (6%), steatosis only (3.3%) and cirrhosis (2%). In 24 cases (16%) one or more viral antigens (HBsAg, HBcAg and Delta Ag) were detected in hepatocytes by immunohistochemical methods. Three liver specimens revealed epithelioid cell granulomas, but in no case was birefringent material identified in the liver. AH and CAH were more frequent in the group of livers with viral antigens. Our histological and immunohistochemical data confirm the current opinion that, in most cases, liver pathology in drug addicts has a viral aetiology, while no significant pathogenic role has been identified for drugs, alcohol or contaminants. Recurrent infections by one or several viruses possibly explain the relatively peculiar histological features of hepatitis in drug addicts.

Delta agent infection in acute hepatitis and chronic HBsAg carriers with and without liver disease.

Hepatitis B virus (HBV) is a major cause of chronic liver disease in southern Italy. In the same area superinfection with the delta agent is endemic. To assess the prevalence of delta infection in a large population of patients with acute and chronic HBV related liver disease and to look for differential features among delta infected and uninfected subjects sera from 592 consecutive HBsAg positive patients were tested for the delta/anti-delta system by RIA. In no case was delta Ag found in serum. The prevalence of anti-delta was low in acute hepatitis (6.6%) and in asymptomatic carriers (6.4%) but raised in chronic active hepatitis with or without cirrhosis (52.3%). A decrease in frequency of anti-delta was seen in inactive cirrhosis (38.8%) and in hepatocellular carcinoma (11.9%). A younger mean age of delta-infected subjects was observed in each type of chronic liver disease. Our data confirm that delta agent superinfection is definitely associated with severe chronic active liver disease. The difference in age between anti-delta positive and negative patients suggests that delta infection accelerates the natural history of HBV related liver disease.

The role of the anion gap in detecting and managing mixed metabolic acid-base disorders

Mixed metabolic-respiratory acid-base disorders may be diagnosed when the respiratory compensation for a primary metabolic acidosis or alkalosis is inappropriate or when there is inappropriate metabolic compensation for a primary respiratory disorder. The magnitude of the primary change in HCO3 concentration (in metabolic disorders) defines the limits of compensation. We emphasized the importance of the equality of the increment in the anion gap (delta AG) and the decrement in the serum bicarbonate concentration (delta HCO3) in diagnosing a simple high AG metabolic acidosis. The close relationship between these two changes in simple high AG acidoses is reviewed. When the delta HCO3 is greater than the delta AG, we suggest that a mixed high AG and hyperchloraemic acidosis is present. Other possible interpretations of these chemical changes are discussed. When the delta HCO3 is less than the delta AG, a mixed metabolic alkalosis and metabolic acidosis is likely to be present, but other additional explanations of this combination are also reviewed. Thus, guidelines are presented as a basis for the use of the delta AG and delta HCO3 for diagnosing and managing mixed metabolic acid-base disorders.

Delta hepatitis agent: structural and antigenic properties of the delta-associated particle.

Delta agent (delta) was serially passaged to a second and third hepatitis B surface antigen (HBsAg) carrier chimpanzee, using as inoculum the peak delta antigen (delta Ag) serum of an animal previously infected with human serum. The characteristics of serially transmitted delta Ag were similar to those described in first-passage animals. It was consistently detected before the development of anti-delta, in association with a 35- to 37-nm subpopulation of HBsAg particles and a unique low-molecular-weight (5.5 X 10(5)) RNA. RNase susceptibility of the delta-associated RNA and release of delta Ag activity upon treatment of delta-associated particles with detergent revealed that this particle is organized into a virion-like form with the RNA and delta Ag as internal components within a coat of HBsAg. Surface determinants of the delta-associated particle other than HBsAg were not detected by radioimmunoprecipitation experiments, using sera of humans and chimpanzees convalescent from delta hepatitis. The HBsAg-associated particle is the "candidate agent" of delta hepatitis.

δ antigen in hepatitis B: Immunohistology of frozen and paraffin-embedded liver biopsies and relation to HBV infection

The finding that the recently described hepatitis B (HB)-associated delta antigen (delta Ag) is preserved in pronase-treated, formalin-fixed paraffin sections allowed a combined prospective and retrospective immunohistological study of its occurrence in 571 liver biopsies. Among 116 frozen biopsies (69 HBAg seropositive, 47 HBAg seronegative) and 455 paraffin-embedded biopsies (296 HBAg seropositive, 159 HBAg-negative), delta Ag was found in 10 HBAg seropositive patients. With the exception of 1 patient with chronic persistent HB, all had chronic-active HB and none had acute HB; 5 patients were i.v. drug abusers. In follow-up biopsies, the delta Ag persisted with HBsAg for as long as 6 years. The expression of delta Ag showed similarities to the HBcAg system including nuclear localization, mixed nuclear cytoplasmic expression, and coexistence with anti-delta in blood. The findings are compatible with the hypothesis that delta Ag represents a transmissible, defective viral agent which requires HBV as a helper and may modulate, but not terminate, ongoing HBV infection.

The hepatitis B virus-associated delta antigen: isolation from liver, development of solid-phase radioimmunoassays for delta antigen and anti-delta and partial characterization of delta antigen.

The delta antigen-antibody (delta Ag/anti-delta) system is associated with type B hepatitis in man. The delta Ag was extracted and solubilized by sonication and treatment with 6 M GuHCl from the nuclei of human liver obtained at autopsy of a delta-positive subject. A microtiter solid-phase radioimmunoassay was developed and used for the partial characterization of delta Ag. Equilibrium centrifugation, gel filtration and chemical and enzymatic treatments revealed delta Ag to be a protein of approximately 68,000 m.w. with a buoyant density of 1.28 g/cm3 in CsCl, stable to heat, acid, nucleases, and glycosidases, and inactivated by alkali and proteases. A standard preparation of delta Ag was used to develop a blocking solid-phase radioimmunoassay for anti-delta.