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CRISPR/dCas9 in genome visualisation. (A) dsDNA detection. dCas9 binds to PAM and sgRNA. dCas9 is fused to a single fluorescent protein or conjugated to multiple fluorescent proteins through Sun-Tag. (B) RNA detection. As in A., but the PAM is extended by an oligonucleotide (PAMmer). More information on the graphical elements is given in the box within the figure.
Source publication
Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme...
Citations
... Once the pg-RNA is released to the cytoplasm, it is soon encapsidated inside the nucleocapsid with the HBV polymerase and reversetranscribed into minus-strand DNA. Finally, the plus-stranded DNA is synthesized to form the partially double-stranded relaxed circular DNA (rcDNA); at this step of viral replication, the matured nucleocapsid can be either recycled to the nucleus of the hepatocyte for amplification/recycling of cccDNA or enveloped by the envelope proteins and excreted from cells to infect naïve cells [10][11][12]. The current antiviral therapies (NUC and interferon-alpha (IFN-a) pegylated or non-pegylated) reduce the viral loads of chronically infected individuals. ...
Introduction:
The burden of chronic hepatitis B virus (HBV) results in almost a million deaths per year. The most common treatment for chronic hepatitis B infection is long-term nucleoside analogs (NUC) or one-year interferon-alpha (pegylated or non-pegylated) therapy before or after NUC therapy. Unfortunately, these therapies rarely result in HBV functional cure because they do not eradicate HBV from the nucleus of the hepatocytes, where the covalently closed circular DNA (cccDNA) is formed and/or where the integrated HBV DNA persists in the host genome. Hence, the search continues for novel antiviral therapies that target different steps of the HBV replication cycle to cure chronically infected HBV individuals and eliminate HBV from the liver reservoirs.
Areas covered:
The authors focus on capsid assembly modulators (CAMs). These molecules are unique because they impact not only one but several steps of HBV viral replication, including capsid assembly, capsid trafficking into the nucleus, reverse transcription, pre-genomic RNA (pgRNA), and polymerase protein co-packaging.
Expert opinion:
Mono- or combination therapy, including CAMs with other HBV drugs, may potentially eliminate hepatitis B infections. Nevertheless, more data on their potential effect on HBV elimination is needed, especially when used daily for 6-12 months.
... 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. ...
Objectives
A 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.
Design
Reference 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.
Results
Hirt 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.
Conclusions
We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
... 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). ...
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. This article is protected by copyright. All rights reserved
In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.
Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid, which must be replicated using the host cell system. It causes large-scale infectious diseases and poses serious threats to the health, social well-being, and economic conditions of millions of people worldwide. Therefore, there is an urgent need to develop novel strategies for accurate diagnosis of virus infection to prevent disease transmission. Quantum dots (QDs) are typical fluorescence nanomaterials with high quantum yield, broad absorbance range, narrow and size-dependent emission, and good stability. QDs-based nanotechnology has been found to be effective method with rapid response, easy operation, high sensitivity, and good specificity, and has been widely applied for the detection of different viruses. However, until now, no systematic and critical review has been published on this important research area. Hence, in this review, we aim to provide a comprehensive coverage of various QDs-based virus detection methods. The fundamental investigations have been reviewed, including information related to the synthesis and biofunctionalization of QDs, QDs-based viral nucleic acid detection strategies, and QDs-based immunoassays. The challenges and perspectives regarding the potential application of QDs for virus detection is also discussed.
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 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 analyzed and summarized, and future development directions are proposed.
