HBV Elimination 101 Brochure Now in Russian: Ликвидация гепатита В (краткое описание политики)

Recently Published Protocols

  • Fluorescent-bait labelling for the ex vivo detection of HBV antigen-specific B cells

    Fluorescently conjugated antigen-bait systems have been extensively used to identify antigen-specific B cells and probe humoral immunity across different settings. Using this principle, HBV antigens are used to bind the B cell receptor (BCR), permitting antigen-specific B cell detection by flow cytometry1,2. Fluorochromes can either be attached covalently via chemical conjugation to the antigen or attached non-covalently by biotinylating the antigen. Dual-staining antigen-baits (where an antigen is directly conjugated to two distinct fluorochromes) have now been used to identify HBsAg- and HBcAg-specific B cells with a high degree of reliability and specificity3. This system can be used to detect and characterise cells ex vivo or adapted to isolate antigen-specific cells using fluorescence-activated cell sorting.
    30 April, 2021
  • Tetramer enrichment for HBV-specific CD8+ T cells

    The frequency of HBV-specific CD8+ T cells can be very low, depending on the specificity. Therefore, they cannot always be detected by conventional ex vivo tetramer staining using 106 PBMCs. Tetramer-based magnetic enrichment enables the detection of these rare virus-specific CD8+ T-cell population by concentration of tetramer-labeled cells. Additionally, if an ex vivo response by conventional tetramer staining can be detected, this enrichment approach can increase the frequency of tetramer-positive cells for the following analysis.
    03 July, 2020
  • A fluorescent in situ hybridization (FISH) assay for detection of HBV DNA in cell culture models

    • Although a plethora of knowledge on the molecular life cycle of Hepatitis B Virus has been gained by utilizing classical methods, they can only inform on the average level of the tested molecule without subcellular or histological context1.
    • Here we describe a modified FISH protocol that allows direct visualization of HBV minus and plus strand DNA in cell culture models (e.g. HepAD38, HepG2-NTCP)2. It can be coupled with immunofluorescence staining of viral or host proteins or any other fluorescent tagging system which could illuminate numerous aspects of virus-host interactions.
    • Our protocol is modified from the ViewRNA ISH cell assay (Thermo Fisher). It is based on a branched DNA signal amplification scheme in which target sequences are hybridized to a series of synthetic oligonucletodies (probe set, preamplifier, amplifier and label probe) and realizes high sensitivity and low background. We found that with some modifications, it also works well for HBV DNA.
    19 June, 2020
  • HBV Ribonuclease H assay resolved by denaturing PAGE

    • This assay characterizes the products of HBV RNase H activity on a DNA:RNA heteroduplex.
    • Products are resolved by denaturing PAGE to assess specificity of cleavage and effects of inhibitory compounds on the enzyme
    06 June, 2019
  • HBV Ribonuclease H FRET Assay

    • This assay detects the activity of HBV RNaseH using fluorescence resonance energy transfer
    • The ribonuclease H (RNaseH) substrate is an RNA DNA heteroduplex in which an RNA oligonucleotide contains a fluorophore and the complementary DNA oligonucleotide contains a fluorescence quencher. Catalytic activity of the RNaseH is measured via an increase in fluorescence stemming from release of the fluor upon degradation of the RNA
    • Inhibition of RNaseH activity can be determined by comparing the change in fluorescence over time between reactions with and without inhibitors
    06 June, 2019
  • A Southern Blot Assay for Detection of HBV cccDNA from Cell Cultures

    • As an essential component of HBV life cycle, the viral covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs (1, 2).
    • HBV cccDNA is responsible for the establishment of viral infection and persistence. Elimination of cccDNA is the ultimate goal to achieve a cure of hepatitis B (3).
    • Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt DNA extraction method; and (2) HBV cccDNA detection by Southern blot analysis.
    • Southern blot remains the “gold standard” technique for cccDNA detection, as it can separate the cccDNA from protein-free (aka deproteinized) relaxed circular DNA (rcDNA) through electrophoresis.
    • This protocol was originally published in Methods in Molecular Biology: Antiviral Methods and protocols (4).
    03 June, 2019
  • Modeling HBV infection and therapy in immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) fumarylacetoacetate hydrolase (FAH) knockout mice with human chimeric liver

    • Besides human, only a few species have been reported to be permissive for Hepatitis B virus (HBV) infection(1-3). Human liver chimeric mouse models have been developed to overcome these limitations.  Currently, three mouse models are available for modeling HBV replication and therapy: urokinase-plasminogen activator (Alb-uPA), fumarylacetoacetate hydrolase knockouts (FAH-/-), and herpes simplex virus type 1 thymidine kinase (TK) transgenic mice(4-9). In these strains, transplanted human hepatocytes have a selective growth advantage. However, FAH-/- and TK transgenic mice provides a good alternative to the fertility defect of uPA mice(10).
    • Knockout of FAH results in the hepatic accumulation of toxic tyrosine metabolic intermediates and subsequent death of mouse hepatocytes which can be controlled by administration/withdrawal of 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC)(4, 11, 12). Here we describe a detailed protocol about 1) maintenance and genotyping of NRG-FAH-/- mice, 2) injection of human hepatocytes, 3) NTBC drug cycle, 4) human albumin, and 5) HBV infection and detection.
    • This protocol was originally published in Hepatitis B Virus: Methods and Protocols (see reference 12)
    25 May, 2019
  • Hepatitis B Virus Polymerase Epsilon RNA Binding Assays

    • HBV polymerase activity is assayed by measuring its ability to bind viral RNA at a secondary-structure motif, the epsilon (e) stem-loop structure.
    • HBV polymerase is expressed in cell culture to yield its binding-active form and RNA may be supplied in culture or in vitro [1].
    • This assay provides a method for evaluating levels of RNA binding by the HBV polymerase, and may include inhibitors or mutant polymerase proteins.
    • This protocol was originally published in Hepatitis B Virus: Methods and Protocols (see reference 1).
    24 May, 2019
  • In Vitro Hepatitis B Virus Polymerase Priming Assay

    • HBV polymerase activity is assayed by measuring the first step in DNA synthesis, protein priming, wherein the polymerase becomes nucleotide-labelled.
    • This in vitroassay uses a plasmid-based expression system performed in cell culture, producing a chaperone-bound, epsilon-dependent polymerase [1].
    • Priming levels are determined by supplying radionucleotides, and measuring autoradiography after polyacrylamide gel electrophoresis.
    • This assay provides a direct test of effects on priming by HBV polymerase inhibitors, and provides a method to screen for functional mutants.
    • An additional assay uses the enzyme Tdp2, which is able to remove, and to allow subsequent visualization of primed products from HBV polymerase.
    • This protocol was originally published in Hepatitis B Virus: Methods and Protocols (see reference).
    23 May, 2019
  • HBV and HDV infection in uPA/SCID mice with humanized livers

    • This protocol describes the infection of liver humanized mice (here UPA/SCID mice) with hepatitis B and /or hepatitis D viruses.
    • We also address the characteristics of infection kinetics and the factors influencing viral spread in humanized mouse livers.
    • The protocol also includes the analysis of viral loads in mouse blood by qPCR to monitor the infection status.
    21 May, 2019