Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges

Bustamante-Jaramillo, Luisa; Fingal, Joshua; Blondot, Marie-Lise; Rydell, Gustaf E.; Kann, Michael

doi:10.3390/v14030557

Cited by 6 publications

(7 citation statements)

References 128 publications

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“…In a multi-step process involving host DNA replication and repair factors the rcDNA is then converted into the episomal cccDNA form (Figure 3a). For an in-depth discussion of cccDNA biology the reader is referred to recent reviews from Bustamante-Jaramillo et al (2022) and Nassal (2015).…”

Section: The Hbv Core Proteinmentioning

confidence: 99%

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Hepatitis B virus movement through the hepatocyte: An update

Prange

2022

Biology of the Cell

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Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3 kb in size is one of the smallest viral genomes known. HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids, and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic shuttling transport, secretory, and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture-based, HBV in vitro-replication and in vitro-infection systems.

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Section: The Hbv Core Proteinmentioning

confidence: 99%

“…For an in‐depth discussion of cccDNA biology the reader is referred to recent reviews from Bustamante‐Jaramillo et al. (2022) and Nassal (2015).…”

Section: The Hbv Core Proteinmentioning

confidence: 99%

Hepatitis B virus movement through the hepatocyte: An update

Prange

2022

Biology of the Cell

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“…However, the limited specificity for cccDNA in a complex mixture of sequence-identical HBV DNA species remains even when using dPCR. Novel methodologies to visualise cccDNA in infected cells are being developed and can complement quantitative analyses and serve to corroborate PCR and SB results 27. Additionally, more research is needed to ascertain the nature of all HBV DNA forms, their role in the life-cycle of HBV and how they are influenced by various manipulations.…”

Section: Discussion and Recommendations For Cccdna Quantificationmentioning

confidence: 99%

“…Novel methodologies to visualise cccDNA in infected cells are being developed and can complement quantitative analyses and serve to corroborate PCR and SB results. 27 Additionally, more research is needed to ascertain the nature of all HBV DNA forms, their role in the life-cycle of HBV and how they are influenced by various manipulations. Reduce HBV RI and rcDNA through means of the DNA extraction ► Extract DNA using Hirt or −PK method to lower RI and rcDNA contained in HBV capsids.…”

Section: Hepatologymentioning

confidence: 99%

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure

Allweiss

Testoni

et al. 2023

Gut

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ObjectivesA major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices.DesignReference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/−PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB.ResultsHirt and −PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA.ConclusionsWe present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.

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“…Therefore, to completely eradicate hepatitis B, cccDNA must be cleared 3,6 . At present, due to the lack of practical, sensitive, and accurate methods to detect cccDNA, research on cccDNA and the development of targeted drugs have progressed slowly 7,8 . On the one hand, cccDNA has the same DNA sequence as all other forms of HBV DNA inside and outside the cell, including relaxed circular DNA (rcDNA), double‐stranded linear DNA (dslDNA), and single‐stranded DNA (ssDNA) 3 .…”

Section: Introductionmentioning

confidence: 99%

Research progress on detection methods for hepatitis B virus covalently closed circular DNA

Sun

Xia

Ouyang

2023

Journal of Viral Hepatitis

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Hepatitis B virus (HBV) infection remains a serious global public health problem, and HBV covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by current treatments and is a major factor in the persistence and recurrence of hepatitis B. Efficient and scientific detection methods are important for clinical monitoring of cccDNA and targeted drug development. Western blotting is the gold standard for the quantitative detection of cccDNA, but it is time‐consuming and complex. In recent years, new detection technologies have been continuously updated. There are new developments and breakthroughs in both next‐generation polymerase chain reaction (PCR) and non‐PCR methods such as in situ hybridization. Some HBV‐related markers (such as hepatitis B core‐related antigen) have also been shown to be closely related to cccDNA, and they can be used as surrogate markers to indirectly reflect cccDNA content. In this paper, the main detection methods of cccDNA and their improvements are reviewed, the advantages and limitations of these methods are analysed and summarized, and future development directions are proposed.

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Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges

Cited by 6 publications

References 128 publications

Hepatitis B virus movement through the hepatocyte: An update

Hepatitis B virus movement through the hepatocyte: An update

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure

Research progress on detection methods for hepatitis B virus covalently closed circular DNA

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