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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.

While Tunisia is endemic for hepatitis B virus (HBV), a recent large-scale retrospective study, revealed a very low prevalence (2%) of hepatitis Delta virus (HDV) (Yacoubi et al. in J Clin Virol 72:126-132, 2015). All strains were classified within the genotype 1 (HDV-1) as assessed by nucleotide sequencing of the so-called 'R0' region of the genome described previously. In this study, we aimed to determine the full-length genome sequence of HDV isolates in order to fully characterize the HDV strains spreading in Tunisia. Eleven HDV antibody and RNA positive samples were obtained from the 1615 clinical samples previously studied. The whole genome sequence was obtained for 5 strains by sequencing and realignment of four overlapping regions covering the entire genome, followed by extensive phylogenetic analyses. Tunisian sequences segregated together with Turkish and African sequences and showed 60% GC content. Alignment with an HDV-1 consensus sequence revealed that they exhibited several point mutations in different functional domains of the delta proteins that, according to previous studies, might possibly affect their properties. In conclusion, the first full-length genome sequences of Tunisian HDV isolates are provided, isolates which are closely related to Turkish and Sub-Saharan Africa strains, supporting the hypothesis for the spread of HDV-1-strains from Africa via Tunisia to Turkey, before spread to the rest of the world.

Hepatitis delta virus (HDV) is responsible for the most severe form of acute and chronic viral hepatitis. We previously proposed that the Deltavirus genus is composed of eight major clades. However, few sequences were available to confirm this classification. Moreover, little is known about the structural and functional consequences of HDV variability. One practical consequence is the failure of most quantification assays to properly detect or quantify plasmatic HDV RNA. Between 2001 and 2014, 2,152 HDV strains were prospectively collected and genotyped in our reference laboratory by means of nucleotide sequencing and extensive phylogenetic analyses of a 400-nucleotide region of the genome (R0) from nucleotides 889 to 1289 encompassing the 3' end of the delta protein-coding gene. In addition, the full-length genome sequence was generated for 116 strains selected from the different clusters, allowing for in-depth characterization of the HDV genotypes and subgenotypes. This study confirms that the HDV genus is composed of eight genotypes (HDV-1 to HDV-8) defined by an intergenotype similarity >85% or >80%, according to the partial or full-length genome sequence, respectively. Furthermore, genotypes can be segregated into two to four subgenotypes, characterized by an intersubgenotype similarity >90% (>84% for HDV-1) over the whole genome sequence. Systematic analysis of genome and protein sequences revealed highly conserved functional nucleotide and amino acid motifs and positions across all (sub)genotypes, indicating strong conservatory constraints on the structure and function of the genome and the protein., Conclusion: This study provides insight into the genetic diversity of HDV and a clear view of its geographical localization and allows speculation as to the worldwide spread of the virus, very likely from an initial African origin. (Hepatology 2017;66:1826-1841)., (© 2017 by the American Association for the Study of Liver Diseases.)

Hepatitis D virus (HDV) is responsible for fulminant hepatitis and liver failure and accelerates evolution toward cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected patients. To date, treatment relies upon long-term administration of pegylated alpha-interferon with a sustained virological response in 30% of the patients. Very recently, new, promising anti-HDV therapies have been developed and are already being used in clinical trials. HDV RNA viral load (HDVL) monitoring must be an integral part of the management of the infected patients. However, HDV genus is characterized by a high genetic variability into eight genotypes (HDV-1 to -8), and most available in-house or commercial assays are useful for only a limited subset of genotypes. Results of a comparison of the performance of a new kit for HDVL quantification with the consensus in-house assay of the French National Reference Laboratory for HDV developed in 2005 are reported here. A total of 611 clinical samples of all HDV genotypes with various HDVL values, including several consecutive samples over several years from 36 patients, were studied. A specificity, sensitivity, and reproducibility evaluation was conducted using HDV-positive clinical samples, hepatitis A, B, C and E (HAV, HBV, HCV, and HEV, respectively) and HIV mono-infected samples, and the WHO HDV RNA international standard. Overall results were strictly comparable between the two assays (median difference, 0.07 log IU/ml), with high diagnosis precision and capacity. In summary, this new kit showed high performance in detection/quantification of HDVL, regardless of the genotype of the infecting strain used, and seems to be a suitable tool for patient management., (Copyright © 2017 American Society for Microbiology.)

Infection by the hepatitis delta virus (HDV), a satellite of the hepatitis B virus (HBV), increases viral liver disease severity. Its diagnosis is thus vital for HBV-infected patients. HDV-RNA load (HDVL) should be assessed and monitored in plasma using real-time reverse-transcriptase polymerase chain reaction assays. Taking advantage of the recently-developed World Health Organization (WHO) HDV international standard (WHO-HDV-IS), the first international external quality control for HDVL quantification was performed. Two panels of samples were sent to 28 laboratories in 17 countries worldwide. Panel A comprised 20 clinical samples of various genotypes (1, 2, and 5-8) and viral loads, including two negative controls. Panel B, composed of dilutions of the WHO-HDV-IS, allowed the conversion of results from copies/mL into IU/mL for HDVL standardization and interlaboratory comparisons. Comprehensive analysis revealed a very high heterogeneity of assay characteristics, including their technical steps and technologies. Thirteen labs (46.3%) properly quantified all 18 positive samples; 16 (57.1%) failed to detect one to up to 10 samples, and several others underestimated (>3 log IU/mL) HDVL of African genotype strains (1 and 5-8). Discrepancies were mainly attributed to either primers or probe mismatches related to the high genetic variability of HDV and, possibly, to the complex secondary structure of the target genomic RNA. The labs were grouped in four clusters by the statistical analysis of their performances. The best clusters comprised the 17 labs that obtained the expected HDVL values, including five that otherwise failed to quantify one or two samples., Conclusion: The results of this international quality-control study underline the urgent need to improve methods used to monitor HDV viremia and will be instrumental in achieving that goal. (Hepatology 2016;64:1483-1494)., (© 2016 by the American Association for the Study of Liver Diseases.)

Background/aims: Treatment of chronic delta hepatitis is long and difficult and better monitoring is needed., Methods: In this study, hepatitis delta virus (HDV) RNA, hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA were retrospectively quantified in 53 patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis delta. Twenty-one had received 28 courses of 3-5 MU interferon-alpha2b (IFN-alpha2b) thrice weekly for a median of 12.6 months (interquartile range [IQR]: 7.3-31.6), five had received eight courses of 100 mg lamivudine (LAM) daily for 23.6 months (IQR: 8.4-61.5) and 27 were untreated. The controls were 54 untreated, randomly selected, HBeAg-negative chronic hepatitis B patients without delta infection. Quantification of serum HDV RNA, HBsAg and HBV DNA were performed at baseline, during and at the end of treatment and end of follow up., Results: Untreated patients had significantly higher median HBsAg levels than controls (5,872 vs 3,501 IU/ml; P = 0.046), but lower median HBV DNA levels (2.933 vs 6.459 log10 copies/ml; P < 0.001). Median baseline HDV RNA (6.374 log10 copies/ml) was similar in IFN-alpha2b-treated, LAM-treated and untreated patients. At the end of treatment, IFN-alpha2b significantly suppressed in paired measurements HDV RNA (P = 0.012) and HBsAg (P = 0.043), but LAM was inefficient. In IFN-alpha2b-treated patients, HDV RNA became undetectable in five patients within a median of 30 months (IQR: 8-90), followed by a slower decrease in HBsAg., Conclusions: In untreated chronic delta hepatitis, suppressed HBV replication is associated with significantly increased HBsAg serum levels. IFN-alpha2b significantly suppresses both HDV RNA and HBsAg, but LAM has no effect. Long-term IFN-alpha2b treatment (IQR: 1.5-5.0 years) appears necessary for undetectable serum HDV RNA and further treatment is required for HBsAg loss. Monitoring of HDV RNA and HBsAg serum levels in patients with chronic delta hepatitis provides insight during treatment.

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected by hepatitis B virus. Interferon alpha at high doses, although poorly efficient, is the only treatment reported to provide some benefit in chronic hepatitis delta. Pegylated interferon alpha (PEG-IFN) has not yet been evaluated. Treatment is usually monitored by the qualitative detection of HDV-RNA in serum. In this study, safety and efficacy of PEG-IFN were assessed in chronic hepatitis delta, and serum HDV-RNA kinetics were determined using quantitative RT-PCR. Fourteen patients with chronic hepatitis delta received subcutaneous PEG-IFN alpha-2b during 12 months (1.5 microg/kg per week). Serum HDV-RNA was quantified at initiation and during the course of therapy, and during the posttreatment follow-up period, which ranged from 6 to 42 months (median 16 months). PEG-IFN alpha-2b was well tolerated, inducing no serious adverse effect. Sustained biochemical response was obtained in 8 patients (57%). At the end of treatment, 8 patients (57%) had achieved virological response (undetectable HDV-RNA). Sustained virological response throughout the posttreatment follow-up period was observed in 6 patients (43%). HDV-RNA kinetics were predictive of the response: after 3 months of PEG-IFN, HDV-RNA levels were significantly lower in the responders than in the nonresponders group (P=.018). After 6 months of therapy, a negative HDV-RNA was predictive of sustained response (P=.021). In conclusion, this preliminary study indicates that PEG-IFN alpha-2b is safe and efficient for treatment of chronic hepatitis delta. The follow-up of HDV-RNA levels during therapy, which allows the differentiation of various profiles of virological responses, improves treatment monitoring.

Hepatitis delta virus (HDV), in association with hepatitis B virus, is responsible for severe acute and chronic hepatitis. Treatment of the infection relies on the long-term administration of high doses of alpha interferon (IFN), and the treatment efficiency is monitored by the detection of anti-HDV immunoglobulin M and HDV genome in serum. Like the case for other chronic viral infections, HDV genome quantification in serum should be useful for the follow-up of infected patients. The aims of this study were to develop a quantitative assay for the detection of any type of HDV in serum and to evaluate the benefit of HDV RNA quantification for the follow-up of chronically infected patients receiving IFN. A real-time reverse transcription-PCR assay was developed to quantify the HDV RNA load in serum. Its efficacy was evaluated with 160 serum samples, 76 of which were collected from 11 chronically infected patients who were treated with pegylated IFN. The assay was sensitive (100 copies/ml of serum) and efficient for all HDV types, including type 3 and the recently described types 5, 6, and 7. The viral load determinations for treated patients allowed us to identify different profiles of virological responses to IFN therapy with more accuracy than that attainable with the qualitative approach. In conclusion, we have developed a quantitative HDV RNA assay for serum which is adapted to the follow-up of antiviral treatment for patients infected with any HDV type. The assay will help us to understand the natural history of HDV infection and to define guidelines for the management of chronic delta hepatitis.

To study the antiviral efficacy of high doses of alpha 2b interferon (alpha 2b-IFN) for chronic hepatitis D treatment, we used polymerase-chain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The semi-quantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By amplifying dilutions (10(-1)-10(-8)) of an HDV-positive woodchuck liver RNA, we confirmed that exponential amplification efficacy occurred at between 20 and 30 cycles. Positive signals were observed from dilution 10(-2) (gel electrophoresis after 20 cycles of PCR) to dilution 10(-7) (hybridization after 30 cycles of PCR). To characterize the HDV RNA level in sera of 8 patients treated with alpha 2b-IFN (10 MU/3 times a week) for 1 year, we extracted RNA from serum samples taken every 6 months. All samples were amplified in parallel for 20 and 30 PCR cycles. Analysis of HDV cDNA after ethidium bromide/agarose gel electrophoresis and after molecular hybridization (100 times more sensitive than gel analysis), enabled us to grade the signals observed from negative to positive as 1+, 2+, 3+ and 4+, with all results being positive. Three types of evolution of HDV viraemia were evidenced among the 8 treated patients. HDV replication continued to occur at a high level at the 6th and 12th month in 2 patient sera. For 2 other patients, an HDV RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12th month.(ABSTRACT TRUNCATED AT 250 WORDS)