Variable In Vivo Hepatitis D Virus (HDV) RNA Editing Rates According to the HDV Genotype.

Human hepatitis delta virus (HDV) is a small defective RNA satellite virus that requires hepatitis B virus (HBV) envelope proteins to form its own virions. The HDV genome possesses a single coding open reading frame (ORF), located on a replicative intermediate, the antigenome, encoding the small (s) and the large (L) isoforms of the delta antigen (s-HDAg and L-HDAg). The latter is produced following an editing process, changing the amber/stop codon on the s-HDAg-ORF into a tryptophan codon, allowing L-HDAg synthesis by the addition of 19 (or 20) C-terminal amino acids. The two delta proteins play different roles in the viral cell cycle: s-HDAg activates genome replication, while L-HDAg blocks replication and favors virion morphogenesis and propagation. L-HDAg has also been involved in HDV pathogenicity. Understanding the kinetics of viral editing rates in vivo is key to unravel the biology of the virus and understand its spread and natural history. We developed and validated a new assay based on next-generation sequencing and aimed at quantifying HDV RNA editing in plasma. We analyzed plasma samples from 219 patients infected with different HDV genotypes and showed that HDV editing capacity strongly depends on the genotype of the strain.

Risk Factors for Delta Hepatitis in a North American Cohort: Who Should Be Screened?

INTRODUCTION: The American Association for the Study of Liver Diseases recommends hepatitis D virus (HDV) screening in certain high-risk groups; however, the effectiveness is unknown. METHODS: A study of North American patients with hepatitis B (HBV) referred to the NIH was performed to identify risk factors associated with HDV infection. Active HDV was "confirmed" by serum HDV RNA or histologic HDV antigen staining. RESULTS: Six hundred fifty-two were studied, of which 91 were HDV "confirmed." Independent risk factors for HDV included: intravenous drug users, HBV-DNA <2,000 IU/mL, alanine aminotransferase >40 U/L, and HDV endemic country of origin. DISUSSION: North American patients with HBV and significant risk factors should be screened for HDV.

High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.

BACKGROUND: Chronic hepatitis delta virus (HDV) infection causes severe liver disease which often leads to cirrhosis and hepatocellular carcinoma (HCC). Aim of this study was to establish the disease severity and prognostic factors for disease outcome by analysing frequencies of clinical events and their correlation with baseline virological and biochemical parameters as well as interferon and nucleos(t)ide analogue treatment choice. METHODS: We studied a single-centre cohort of 49 anti-HDAg-positive patients with HBsAg persistence for at least 6 months. Virological and biochemical parameters, interferon and nucleos(t)ide analogue treatment choice as well as clinical events during follow-up were analysed by retrospective chart review (mean follow-up time 3 years, range 0.25-7.67 years). RESULTS: Severe clinical events occurred in 11/49 hepatitis D patients, including HCC (8/49), death (8/49) or liver transplantation (2/49). HCCs only occurred secondary to liver cirrhosis and their event rates in this cohort of hepatitis D patients did not differ from a matched HBV mono-infected cohort with comparable frequency of liver cirrhosis. A stepwise multivariate logistic regression revealed low platelet count (p = 0. 0290) and older age (p = 0.0337) correlating most strongly with overall clinical events, while serum HDV RNA positivity at baseline did not correlate with any clinical outcome. Interferon-free but not nucleos(t)ide analogue-free patient care correlated with the occurrence of HCC at logistic regression, although only 3/18 interferon-treated patients demonstrated repeatedly negative HDV PCR results post therapy. CONCLUSIONS: Our data indicate that progressive liver disease at baseline plays a major role as predictive factor for overall clinical outcome of hepatitis D patients. In particular, HCC risk may not be underestimated in hepatitis D virus RNA negative hepatitis D patients with advanced liver fibrosis.

Development and performance of prototype serologic and molecular tests for hepatitis delta infection.

Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.

The hepatitis delta genotype 8 in Northeast Brazil: The North Atlantic slave trade as the potential route for infection.

Hepatitis Delta virus (HDV) is not well known, even though HDV and Hepatitis B virus (HBV) co-infection leads to severe forms of acute and chronic liver diseases. HDV is endemic in the Western Amazon region. Recently, the HDV genotype 8 was found in chronic patients followed at the center for liver studies in the Northeast Brazil, Maranhão. Previous studies suggested that this genotype was introduced in Maranhão during the slave trade. The presence of HDV in that study, which was done outside the Amazon region, led us to investigate whether the virus is found infecting individuals in other regions of Maranhão as well. Thus, we screened ninety-two HBsAg positive individuals from five Municipalities of Maranhão for anti-HD antibody and eight were found positive (8.7%). These eight positive individuals were submitted to polymerase chain reaction (PCR) to investigate active HDV infection. Half of them were positive for a fragment sequence of the delta antigen; their sequence samples were submitted to genotype characterization by phylogenetic analysis. All sequences clustered in a unique branch of the tree separated from the other branch described in Africa. Our study confirmed the presence of HDV-8 in Maranhão. These infected individuals had no evidence of contact with African people. Furthermore, we found individuals infected with HDV-8 in two more different municipalities. More studies like ours are urgent because the co-infection HBV/HDV is more difficult to treat. Identification of the endemic regions and implementation of healthy policies for preventing this infection are urgent in this region.

The adventure of delta.

The hepatitis D virus (HDV) is unique in animal virology. It has a circular RNA genome that is the smallest of human viruses, requires the HBsAg capsid of the hepatitis B virus to assembly into infectious virions, parasitizes the transcriptional machinery of the host by hijacking cellular RNA polymerases to replicate its RNA genome and is replicated by a rolling circle mechanism unknown to mammalian cells. Hepatitis D is ubiquitous but prevalence varies throughout the world. It is the most severe form of chronic viral liver disorder; carriers of HBsAg superinfected by the HDV are the major victims and the reservoir of the infection. In the last 20 years vaccination against the hepatitis B virus (HBV) has decreased the circulation of HDV in industrialized countries; nevertheless hepatitis D is returning to Western Europe through immigration from HDV endemic areas. Hepatitis D is being rediscovered in the developing world. It has a significant medical impact on areas of Africa, Asia and South America where the partner HBV is not controlled; Pakistan and Mongolia appear to be worldwide the areas with the highest prevalence of the disease. A major obstacle in treatment is that the virus has no replicative function of its own to be targeted by antivirals. Peg-Interferon remains the mainstay of treatment. New strategies are explored to prevent entry of the virion into hepatocytes by blocking the cellular HBsAg receptor or preventing the prenylation process of the large-delta antigen necessary for the assembly of the HDV particle.

Analysis on the clinical features of 507 HDV-infected patients.

The objective of this study was to analyze the clinical feature of hepatitis delta virus (HDV)-infected patients and to discuss the pathological mechanism of hepatitis D. A total of 507 patients with hepatitis due to the infection of HDV were included. The incidence rates of various hepatitis subtypes, the sequelae, the clinical manifestation, the hepatic function, and the hepatic virus makers for all the 507 patients were analyzed statistically. A cohort of 213 patients with hepatitis B, who were also HDV free, served as the control. HDV infection significantly contributed to the increased incidence rate and mortality of severe hepatitis (SH) and cirrhosis (P < 0.01). HDV was also associated with higher incidence rates of hemorrhage in the gastrointestinal tract, abdominal ascites and hepatic encephalopathy, repetitive augmentation of alanine transaminase, and its enhanced magnitude (P < 0.01 or 0.05). The major liver function changes in patients with SH or chronic serious hepatitis was significant compared to the control (P < 0.01). Within the HDV(+) category, HBeAg(-) expression was significantly higher in the HBV DNA(-) group than the HBV DNA(+) group (P < 0.01). The expression of HDAg(+) HBeAg(-) in acute hepatitis, SH, and cirrhosis was significantly higher than that of HDAg(+) HBeAg(+) (P < 0.01 or P < 0.05). The HDV infection was closely associated with the development and prognosis of chronic serious hepatitis, SH, and cirrhosis. HDV infection could inhibit the HBV DNA replication or the HBcAg expression. The direct cytotoxicity of HDV might be the leading pathological factor in AH. HDV might play a major role in the deterioration and chronicization of HDV-co-infected hepatitis B and was responsible for the increased mortality of HBV/HDV patients.

Characterization of hepatitis delta virus in sub-Saharan Africa.

Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV), and infection with this virus aggravates acute and chronic liver disease. While HBV seroprevalence is very high across sub-Saharan Africa, much less is known about HDV in the region. In this study, almost 2,300 blood serum samples from Burkina Faso (n=1,131), Nigeria (n=974), Chad (n=50), and the Central African Republic (n = 118) were screened for HBV and HDV. Among 743 HBsAg-positive serum samples, 74 were positive for HDV antibodies and/or HDV RNA, with considerable differences in prevalence, ranging from <2% (pregnant women from Burkina Faso) to 50% (liver patients from Central African Republic). HDV seems to be much more common in chronic liver disease patients in the Central African Republic (CAR) than in similar cohorts in Nigeria. In a large nested mother-child cohort in Burkina Faso, the prevalence of HDV antibodies was 10 times higher in the children than in their mothers, despite similar HBsAg prevalences, excluding vertical transmission as an important route of infection. The genotyping of 16 full-length and 8 partial HDV strains revealed clade 1 (17/24) in three of the four countries, while clades 5 (5/24) and 6 (2/24) were, at least in this study, confined to Central Nigeria. On the amino acid level, almost all our clade 1 strains exhibited a serine at position 202 in the hepatitis D antigen, supporting the hypothesis of an ancient African HDV-1 subgroup. Further studies are required to understand the public health significance of the highly varied HDV prevalences in different cohorts and countries in sub-Saharan Africa.

Resolution of chronic hepatitis delta infection after five years of peginterferon-adefovir: lessons from a case report.

There is still some controversy about the treatment of hepatitis delta virus (HDV) infection and the treatment endpoints. A 48-year-old patient was treated with a combination of peginterferon-α and adefovir, and HDV RNA clearance occurred after 3 years of treatment. However, treatment was continued until HBs antigen (Ag) seroconversion, which occurred after 5 years of therapy. One year after the end of the treatment, the patient was still HBs Ag and HDV RNA negative. This case report suggests that combined peginterferon-α and adefovir may be effective in treating HDV infection and, if given over a longer period, may result in hepatitis B surface antigen (HBsAg) seroconversion. It highlights the interest of using HBsAg quantification associated with a sensitive RT-PCR approach for monitoring the treatment of chronic hepatitis delta. HBsAg seroconversion, or at least significant decrease, could be a more relevant endpoint than HDV RNA undetectability for discontinuing HDV treatment and preventing the occurrence of virological relapses.

Hepatitis delta virus diagnosis.

The first step in the diagnosis of hepatitis delta virus (HDV) infection is testing HBsAg-positive individuals for the antibody to the HD antigen (anti-HD).In anti-HD-positive patients, the next step is testing for HDV RNA in serum to determine whether the antibody reflects an ongoing active HDV infection (HDV-RNA-positive) or represents a serologic scar to past HDV infection (HDV-RNA-negative). In the HDV-positive individual with liver disease, it is critical to distinguish acute HDV/hepatitis B virus (HBV) coinfection from chronic HDV superinfection in HBsAg carriers; the course, prognosis, and management of the two conditions are different. The differential diagnosis can be achieved through the scrutiny of the battery of HDV and HBV markers, which combine in patterns characteristic for each condition.Standardized competitive and µ-capture commercial assays are available to determine the IgG and IgM antibody to HDV. Several in-house assays were developed to determine HDV RNA in serum; the sensitivity threshold of current polymerase chain reaction-based assays is 10 copies of HDV RNA/mL. Unfortunately, HDV-RNA assays are not yet standardized and the results from different laboratories often are not comparable due to different sensitivities. The development of an international reference HDV-RNA standard remains an unmet diagnostic need.

A case of quadruple viral infections and elevated aminotransferase activities.

A 40-year-old man with human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infection was referred for evaluation of abnormal liver enzyme activities. The patient was maintained on antiretroviral therapy for HIV as well as medication to suppress HBV and had previously undergone treatment for HCV with durable sustained virologic response. The patient was clinically well without any symptoms or evidence of liver decompensation. Laboratory findings were notable for aminotransferase activities in the 200 to 225 U/L range that had been persistent for several months. An extensive workup for the etiology of the aminotransferase elevation ensued. Imaging studies showed no evidence of biliary obstruction. Serology revealed negative autoantibodies, negative serum HCV-RNA, and low level HBV-DNA by polymerase chain reaction. Further testing revealed positive hepatitis delta virus (HDV) antibody and positive HDV RNA in the serum. A percutaneous liver biopsy was performed to further elucidate the cause of the elevated aminotransferase activities. Based on histology, serology, and clinical presentation, a diagnosis of chronic HDV infection was made. HDV infection should be considered in patients with known chronic viral hepatitis B with low viral load, who present with worsening liver function or elevation in aminotransferase activities.

HDAg-L variants in covert hepatitis D and HBV occult infection among Amerindians of Argentina: new insights.

BACKGROUND: Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) polymerase chain reaction (PCR). OBJECTIVES: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. STUDY DESIGN: Forty-six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110-195) and HDAg-L (aa110-214) was performed. RESULTS: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc-only positive, 2 of them identified as cases of occult hepatitis B infection (OBI). The 3 cases were HBeAg-negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 - reportedly known to promote in vitro an unsuitable interaction with HBsAg - were observed. CONCLUSIONS: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.

Seroprevalence of antibody to HDV in Nigerians with hepatitis B virus-related liver diseases.

OBJECTIVE: Hepatitis D Virus (HDV) infection has been reported to be declining in some geographical areas. In order to ascertain the current status of HDV infection in Nigeria, a study of patients with hepatitis B virus (HBV)-related liver diseases was undertaken to determine the sero-prevalence ofanti-HDV. METHOD: This was a prospective, cross-sectional study in which all consecutive patients with liver disease who tested positive for Hepatitis B surface antigen (HBsAg) were also tested for antibody to HDV. RESULT: Ninety six patients with various forms of HBV-related liver diseases participated in the study (acute hepatitis 8.3%, asymptomatic infection 15.6%, chronic hepatitis 3.1%, liver cirrhosis 21.9% and primary liver cell carcinoma 51.0%). Anti-HDV was demonstrated in 12 patients (12.5%). In patients with acute hepatitis and asymptomatic infection the prevalence was 4.3% while in patients with chronic hepatitis, liver cirrhosis and primary liver cell carcinoma, the prevalence was 15%. CONCLUSION: HDV still contributes to significant morbidity and mortality in HBV-related liver diseases in Nigeria. There is urgent need for larger studies on a national scale to accurately appraise the public health importance of this infection.

[Expression of hepatitis delta antigen Inner Mongolian strain in prokaryotic cell and analysis of its antigenicity].

OBJECTIVE: To obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity. METHODS: Hepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA. RESULTS: Comparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistency was good (100%). CONCLUSION: EIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.

Search for hepatitis delta virus (HDV) infection in hepatitis C patients in north-eastern Poland. Comparison with anti-HDV prevalence in chronic hepatitis B.

Hepatitis C virus and hepatitis D virus have been shown to suppress HBsAg synthesis. Thus it is possible that HDV infection occurs despite the lack of detectable HBsAg. The aim of our study was to (a) determine the prevalence of HDV infection in patients with chronic hepatitis C (b) compare it with the prevalence of HDV infection in HBsAg positive patients with hepatitis B. The study group consisted of 51 chronic hepatitis C patients, 30 HIV infected drug addicts (27 of them were also positive for anti-HCV) and 102 hepatitis B patients. The participants were tested for anti-HDV, anti-HCV and HBsAg. All anti-HCV positive patients were negative for anti-HDV. Four individuals with anti-HDV belonged to hepatitis B group and constituted 3.9% of all HBsAg positive subjects. We conclude that (a) there is currently no evidence of HDV infection among HCV infected patients in our region (b) hepatitis delta infection is rare in north-eastern Poland.