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.

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.

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.

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).

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.

Development of a novel system to study hepatitis delta virus genome replication.

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (deltaAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of deltaAg species that are nonfunctional or even inhibitory. A replication system was devised where all deltaAg was conditionally provided from a separate and unchanging source.

Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs.

The genome of hepatitis delta virus (HDV) is a small single-stranded circular RNA that is replicated via RNA-directed RNA synthesis. This makes use of a host RNA polymerase, probably pol II, that normally transcribes DNA templates. In vivo, the host polymerase can initiate replication from transfected linear RNAs using intramolecular template-switching.

Features affecting the ability of hepatitis delta virus RNAs to initiate RNA-directed RNA synthesis.

In models of the replication of human hepatitis delta virus (HDV) RNA, it is generally assumed that circular RNAs are the only templates. However, noncircular HDV RNAs are also produced during replication, and it is known that replication can be initiated by transfection with noncircular RNAs. Therefore, strategies were devised to determine the relative ability of different HDV RNA species to initiate RNA replication.