Sera of Individuals Chronically Infected with Hepatitis B Virus (HBV) Contain Diverse RNA Types Produced by HBV Replication or Derived from Integrated HBV DNA.

This study aimed to better characterize the repertoire of serum hepatitis B virus (HBV) RNAs during chronic HBV infection in humans, which remains understudied. Using reverse transcription-PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), RNA-sequencing, and immunoprecipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA, including 5'-HBV-human-3' RNAs (integrant-derived RNAs [id-RNAs]) and 5'-human-HBV-3' transcripts, as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pregenomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) the area of HBV positions 1531 to 1739 had very high RNA read coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) the vast majority of rd-RNAs and pgRNA were associated with HBV virions but not with unenveloped capsids, exosomes, classic microvesicles, or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum relaxed circular DNA (rcDNA) and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms.

N-Methyladenine Modification of Hepatitis Delta Virus Regulates Its Virion Assembly by Recruiting YTHDF1.

Hepatitis delta virus (HDV) is a defective satellite virus that uses hepatitis B virus (HBV) envelope proteins to form its virions and infect hepatocytes via the HBV receptors. Concomitant HDV/HBV infection continues to be a major health problem, with at least 25 million people chronically infected worldwide. N-methyladenine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally, and this modification regulates various biological processes.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets.

Involvement of Innate Immune Receptors in the Resolution of Acute Hepatitis B in Woodchucks.

The antiviral property of small agonist compounds activating pattern recognition receptors (PRRs), including toll-like and RIG-I receptors, have been preclinically evaluated and are currently tested in clinical trials against chronic hepatitis B (CHB). The involvement of other PRRs in modulating hepatitis B virus infection is less known. Thus, woodchucks with resolving acute hepatitis B (AHB) after infection with woodchuck hepatitis virus (WHV) were characterized as animals with normal or delayed resolution based on their kinetics of viremia and antigenemia, and the presence and expression of various PRRs were determined in both outcomes.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies.

Down-regulation of hepatitis delta virus super-infection in the woodchuck model.

Mechanisms mediating clearance of hepatitis delta virus (HDV) are poorly understood. This study analyzed in detail profound down-regulation of HDV infection in the woodchuck model. Super-infection with HDV of woodchucks chronically infected with HBV-related woodchuck hepatitis virus produced two patterns.

Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers.

Five matching sets of nonmalignant liver tissues and hepatocellular carcinoma (HCC) samples from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined by using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV genotype C, while one pair was infected with HBV genotype B.

Structural and nucleic acid binding properties of hepatitis delta virus small antigen.

Aim: To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195 (∆60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization.

Methods: Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ∆60HDAg. Nucleic acid binding properties were investigated by gel retardation assays.

Measurement of Antiviral Effect and Innate Immune Response During Treatment of Primary Woodchuck Hepatocytes.

An estimated 350 million people are chronically infected with hepatitis B virus (HBV), and over one million people die each year due to HBV-associated liver diseases, such as cirrhosis and liver cancer. Current therapeutics for chronic HBV infection are limited to nucleos(t)ide analogs and interferon. These anti-HBV drugs in general reduce viral load and improve the long-term outcome of infection but very rarely lead to a cure.

Hepatitis delta virus: A fascinating and neglected pathogen.

Hepatitis delta virus (HDV) is the etiologic agent of the most severe form of virus hepatitis in humans. Sharing some structural and functional properties with plant viroids, the HDV RNA contains a single open reading frame coding for the only virus protein, the Delta antigen. A number of unique features, including ribozyme activity, RNA editing, rolling-circle RNA replication, and redirection for a RNA template of host DNA-dependent RNA polymerase II, make this small pathogen an excellent model to study virus-cell interactions and RNA biology.

Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection.

Unlabelled: The infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naive adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum.

Capacity of a natural strain of woodchuck hepatitis virus, WHVNY, to induce acute infection in naive adult woodchucks.

Woodchuck hepatitis virus (WHV) is often used as surrogate to study mechanism of HBV infection. Currently, most infections are conducted using strains WHV7 or WHV8 that have very high sequence identity. This study focused on natural strain WHVNY that is more genetically distant from WHV7.

Superinfection with woodchuck hepatitis virus strain WHVNY of livers chronically infected with strain WHV7.

Unlabelled: The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV).

Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.

Unlabelled: This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes A to I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV ribonucleoprotein and differed only by the HBV variant-specific envelope proteins coating the particles. The total HDV yields varied within a 122-fold range.

Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles.

Unlabelled: A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication.

Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks.

Unlabelled: Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood.

Intracellular localization of hepatitis delta virus proteins in the presence and absence of viral RNA accumulation.

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (deltaAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that deltaAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, deltaAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of deltaAg in the presence and absence of replicating and nonreplicating HDV RNAs.

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH).

Assembly of hepatitis B virus envelope proteins onto a lentivirus pseudotype that infects primary human hepatocytes.

This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry.

Immunoadhesins containing pre-S domains of hepatitis B virus large envelope protein are secreted and inhibit virus infection.

Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S.

Two potentially important elements of the hepatitis B virus large envelope protein are dispensable for the infectivity of hepatitis delta virus.

Previous studies have attempted to clarify the roles of the pre-S1 and pre-S2 domains of the large envelope protein of hepatitis B virus (HBV) in attachment and entry into susceptible cells. Difficulties arise in that these domains contain regions involved in the nucleocapsid assembly of HBV and overlapping with the coding regions of the viral polymerase and RNA sequences required for reverse transcription. Such difficulties can be circumvented with hepatitis delta virus (HDV), which needs the HBV large envelope protein only for infectivity.

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.

Restoration in vivo of defective hepatitis delta virus RNA genomes.

The 1679-nt single-stranded RNA genome of hepatitis delta virus (HDV) is circular in conformation. It is able to fold into an unbranched rodlike structure via intramolecular base-pairing. This RNA is replicated by host RNA polymerase II (Pol II).

Action of inhibitors on accumulation of processed hepatitis delta virus RNAs.

Hepatitis delta virus (HDV) replication involves processing and accumulation of three RNA species: the genome, its exact complement (the antigenome), and a polyadenylated mRNA that acts as a template for the small delta antigen (deltaAg), the only protein of HDV and essential for genome replication. In a recently reported experimental system, addition of tetracycline induced synthesis of a DNA-directed source of deltaAg, producing within 24 h a significant increase in accumulation of newly transcribed and processed HDV RNAs. This induction was used here to study the action of various inhibitors on accumulation.

Evolution of hepatitis delta virus RNA genome following long-term replication in cell culture.

Previous studies have defined a novel cell culture system in which a modified RNA genome of hepatitis delta virus (HDV) is able to maintain a low level of continuous replication for at least 1 year, using a separate and limited DNA-directed source of mRNA for the essential small delta protein. This mode of replication is analogous to that used by plant viroids. An examination was made of the nucleotide changes that accumulated on the HDV RNA during 1 year of replication.