This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

1. Production of Recombinant HBV Encoding the Reporter Protein
<ol>
	<li>Preparation of HepG2 cells
	<ol>
		<li>Prepare cell culture medium (Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 100 U/ml nonessential amino acids).</li>
		<li>Plate 4 x 106 HepG2 cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at 37 &#176;C in a humidified 5% CO2 incubator. 
		NOTE: When approximately 4 x 106 HepG2 cells are plated in a 10 cm collagen-coated dish in 10 ml of culture medium, they will be 70-90% confluent the next day.</li>
	</ol>
	</li>
	<li>Transfection
	<ol>
		<li>Transfect HepG2 cells with 5 &#181;g of pUC1.2HBV delta epsilon15 and 5 &#181;g of pUC1.2HBV/NL15 using a transfection reagent as per manufacturer&#39;s instructions.</li>
		<li>The next day, remove the culture medium and add 10 ml of fresh culture medium.</li>
		<li>One week after transfection, transfer the culture medium containing the recombinant HBV to a 50 ml-tube and proceed to step 1.3.1. Add 10 ml of fresh culture medium to the plate containing the transfected cells. 
		NOTE: Production of the recombinant HBV is maintained for 4 weeks. The culture medium containing recombinant HBV can be stored at 4 &#176;C for 1 month.</li>
	</ol>
	</li>
	<li>Purification of recombinant HBV
	<ol>
		<li>Remove cell debris from the culture medium containing recombinant HBV by centrifugation (2,300 x g for 5 min).</li>
		<li>Pass the supernatant through a 0.45 &#181;m membrane filter.</li>
		<li>Add an equal volume of 26% PEG/1.5 M NaCl (polyethylene glycol 6000: 130 g, NaCl, 49 g, 1 ml of 0.5 M EDTA pH 8.0 and 5 ml of 1 M HEPES pH 7.6 in 500 ml) to the culture medium containing recombinant HBV and mix gently. Incubate overnight at 4 &#176;C.</li>
		<li>Centrifuge at 2,300 x g for 20 min at 4 &#176;C.</li>
		<li>Discard the supernatant and dissolve the pellet in 0.5 ml of TNE (10 mM Tris, 50 mM NaCl, 1 mM EDTA).</li>
		<li>Remove the debris by centrifugation at 2,300 x g for 5 min.</li>
		<li>Load 0.5 ml of TNE containing recombinant HBV onto 0.8 ml of 20% sucrose in TNE.</li>
		<li>Centrifuge at 100,000 &#215; g for 3 hr at 15 &#176;C.</li>
		<li>Discard as much of the supernatant as possible, and save the pellet. Resuspend it in 1 ml of serum free DMEM per 40 ml of starting culture medium.</li>
		<li>Incubate overnight at 4 &#176;C.</li>
		<li>Filter through a 0.45 &#181;m filter. Prepare 0.5 ml aliquots and store at -80 &#176;C. 
		NOTE: If reporter protein contamination of the original virus sample is observed, purify the virus by density gradient ultra-centrifugation of 5-30% sucrose in TNE at 100,000 x g for 2 hr, or CsCl density equilibrated centrifugation from 1.1-1.6 g/ml at 150,000 x g for 50 hr.</li>
	</ol>
	</li>
</ol>
2. Infection of Recombinant HBV
<ol>
	<li>Culture HepG2 cells stably expressing NTCP (HepG2/NTCP)15 that are susceptible to HBV infection in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 250 &#181;g/ml G-418 and 100 U/ml nonessential amino acids at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>One day before infection, plate approximately 5 x 104 HepG2/NTCP cells14 into a 96-well collagen coated plate in 0.1 ml of culture medium.</li>
	<li>Thaw recombinant HBV in a 37 &#176;C water bath until there is a small bit of ice remaining in the vial.</li>
	<li>Prepare the medium for infection by combining the following:10 &#181;l of 40% PEG8000 in 1x phosphate buffered saline (PBS), 2 &#181;l of dimethyl sulfoxide (DMSO), 10 &#181;l of recombinant HBV and 78 &#181;l of fresh culture medium per well of a 96-well plate.</li>
	<li>Add 100 &#181;l of recombinant HBV solution to one well of the 96-well plate.</li>
	<li>One day after infection, wash the infected cells 3 times with 300 &#181;l PBS per well to remove the contaminating reporter protein from the virus fraction.</li>
	<li>Incubate infected cells in 200 &#181;l of culture medium containing 2% DMSO for one week or less.</li>
</ol>
3. Analysis
<ol>
	<li>One week after infection, wash the infected cells 3 times with PBS.</li>
	<li>Add 50 &#181;l of lysis buffer to the infected cells.</li>
	<li>Rock the culture plate for 5 min, and then centrifuge at 2,000 x g for 5 min.</li>
	<li>Add 50 &#181;l of the reporter substrate into the luminometer plate.</li>
	<li>Add 20 &#181;l of cell lysate to a luminometer plate containing the reporter substrate. Mix by vortexing briefly.</li>
	<li>Place the plate in the luminometer and initiate reading15.</li>
</ol>

<ol>
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Hepatol. 13 (4), 239-248 (2016).</li><li>Wursthorn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peginterferon+alpha-2b+plus+adefovir+induce+strong+cccDNA+decline+and+HBsAg+reduction+in+patients+with+chronic+hepatitis+B.">Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.</a> Hepatology. 44 (3), 675-684 (2006).</li><li>Stuyver, L. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate.">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS. Chem. Biol. 7 (11), 1848-1857 (2012).</li><li>Nishitsuji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+reporter+system+to+monitor+early+stages+of+the+hepatitis+B+virus+life+cycle.">Novel reporter system to monitor early stages of the hepatitis B virus life cycle.</a> Cancer. Sci. 106 (11), 1616-1624 (2015).</li><li>Sugiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+hepatitis+B+virus+genotypes+on+the+intra-+and+extracellular+expression+of+viral+DNA+and+antigens.">Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.</a> Hepatology. 44 (4), 915-924 (2006).</li><li>Seeger, C., Zoulim, F., Mason, W., Knipe, D., Howley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepadnaviruses.">Hepadnaviruses.</a> Field Virology. Volume 1. , 2185-2221 (2013).</li><li>Moolla, N., Kew, M., Arbuthnot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+elements+of+hepatitis+B+virus+transcription.">Regulatory elements of hepatitis B virus transcription.</a> J. Viral. Hepat. 9 (5), 323-331 (2002).</li><li>Cattaneo, R., Will, H., Schaller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+transcription+in+the+infected+liver.">Hepatitis B virus transcription in the infected liver.</a> EMBO. J. 3 (9), 2191-2196 (1984).</li><li>Enders, G. H., Ganem, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+major+transcripts+of+ground+squirrel+hepatitis+virus:+the+presumptive+template+for+reverse+transcriptase+is+terminally+redundant.">Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant.</a> Cell. 42 (1), 297-308 (1985).</li><li>Haines, K. M., Loeb, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sequence+of+the+RNA+primer+and+the+DNA+template+influence+the+initiation+of+plus-strand+DNA+synthesis+in+hepatitis+B+virus.">The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus.</a> J. Mol. Biol. 370 (3), 471-480 (2007).</li><li>Hirsch, R. C., Lavine, J. 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The authors declare that they have no competing financial interests.

The HBV genome has four primary open reading frames (ORFs) including the core, polymerase, surface and X ORFs (Figure 1). Transcription of these four HBV ORFs is tightly regulated by four promoters: the precore/core promoter containing enhancer II, S1 promoter, S2 promoter and X promoter containing enhancer I18. The 3.5 kb and 3.4 kb mRNAs are translated into PreCore and Core proteins, respectively. The large envelop protein (L) is produced from the largest subgenomic mRNA (2.5 kb...

Chronic infection with hepatitis B virus (HBV) is a major risk factor for chronic liver diseases1. Although current therapeutic strategies are based on nucleotide analogs that inhibit HBV pol function and/or the administration of type I interferon that activates immune responses in infected individuals as well as indirectly suppressing HBV proliferation through interferon-stimulated gene functions2, these treatments cannot eliminate HBV DNA completely3. Moreover, the emergence of HBVs that are resistant to anti-pol agents is of concern4. The administration of combined anti-viral agents directly targeting the different steps of human immunodeficiency virus (HIV) or hepatitis C virus (HCV) life cycle was shown to successfully suppress or eradicate the virus(es). Similar to this idea, the development of anti-HBV agent(s) that act directly on the different stages of the HBV life cycle is important for establishing future HBV therapies.
In general, the establishment of a simple in vitro culture system of the target virus facilitates the development of anti-virus agents. However, there are at least two barriers to the development of in vitro culture systems to screen anti-HBV agents. The first is the lack of a convenient in vitro cell culture system for HBV infection/proliferation. Unlike other viruses, such as HIV and HCV, which are propagated in established cell lines, it is difficult to cultivate HBV in vitro because of experimental limitations including a narrow host range. The use of specific cell culture systems such as the human hepatoma cell line HepaRG, which is susceptible to HBV infection,5,6,7 have been developed to overcome these problems. Moreover, PXB cells, isolated from urokinase-type plasminogen activator transgenic/SCID mice inoculated with primary human hepatocyte (PHH), were shown to be susceptible to HBV infection and replication8. However, HBV replication levels in HepaRG are dependent on the cellular differentiation state after culture, which can cause inconsistent and irreproducible results of HBV infection/replication levels. PXB is commonly used for HBV infection experiments but is limited by its availability. A tetracycline inducible HBV expression cell line, HepAD38, has also been widely used to study HBV replication, but this system only allows evaluation after transcription and not at the entry step of HBV infection9. Recently, the identification of sodium taurocholate cotransporting polypeptide (NTCP) as a functional receptor for HBV has allowed the development of a variable HBV culture system10. Indeed, NTCP expression in non-susceptible hepatocarcinoma cells such as Huh7 and HepG2 enables HBV infection10 and thus, the choice of HBV susceptible cell lines has been expanded, resolving many of the experimental limitations. The second problem is the lack of a simple assay system to evaluate HBV infection and replication. Evaluation of HBV infection is usually conducted by analyzing HBV DNA, RNA and proteins. However, quantification of these virus markers is time consuming, often costly and not always simple. Therefore, the development of a simple assay system, such as using a reporter gene, might overcome problems associated with HBV assay systems.
However, because the genome size that can be packaged into an HBV capsid is limited &#8212; less than 3.7 kb11 &#8212; the size of a reporter gene should be as short as possible. Furthermore, the presence of multiple cis elements scattered throughout the genome, which are essential for viral replication, limits the positions available for insertion of the reporter gene into the genome. Several reports have attempted to insert foreign genes, including HIV-1 Tat, green fluorescent protein, and DsRed, into the HBV genome11,12,13. However, these recombinant HBVs are not useful for screening HBV infection/replication, or for the high-throughput screening of factors affecting HBV infection/replication. This is mainly because of the low productivity of recombinant viruses and the reduced intensity of reporter gene expression caused by inefficient virus production.
To overcome these issues, we constructed a reporter HBV with a high yield of virus production. This virus is highly sensitive for monitoring the early stages of the HBV replication cycle, from entry to transcription. To achieve this, NanoLuc (NL) was chosen as a marker gene because it is a small (171 amino acids) engineered luminescent reporter14. Moreover, NL is approximately 150-fold brighter than firefly or Renilla luciferase, and the luminescent reaction is ATP-independent, suggesting that the false hit rate will be low for high-throughput screening. The production efficiency of the recombinant HBV is approximately 1/5 of the parent HBV, and similar to levels reported for previous HBV recombinant viruses; however, the brightness of NL overcomes virus productivity issues so it can be used for the mass screening of anti-HBV agents.
Screening of anti-HBV agents using primary hepatocytes, HepaRG, HepAD38 and NTCP-transduced hepatocytes might be useful for the screening of anti-HBV agents by conventional method(s). However, the system described here has various advantages such as simple handling, high sensitivity, and low cost for screening. These advantages are suitable for high throughput assays to develop and identify new HBV agents for therapeutic purposes.

<table><tbody><tr><td>Nano-Glo Luciferase Assay Regent</td><td>Promega</td><td>N1110</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>Nacalai tesque</td><td>09367-34&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Thermo Fisher Scientific</td><td>11140050</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>100 mm/collagen-coated dish</td><td>Iwaki</td><td>4020-010</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>L3000001&amp;nbsp;</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 6000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>81255</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 8000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>NaCl</td><td>Nacalai tesque</td><td>31319-45&amp;nbsp;</td><td></td></tr><tr><td>0.5 mol/L EDTA Solution</td><td>Nacalai tesque</td><td>06894-14</td><td></td></tr><tr><td>Tris-HCl</td><td>Nacalai tesque</td><td>35434-21</td><td></td></tr><tr><td>Millex-HP, 0.45 &amp;mu;m, polyethethersulfone, filter</td><td>Merck Millipore</td><td>SLHP033RS</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Collagen coated 96-well plate</td><td>Corning</td><td>NO3585</td><td></td></tr><tr><td>Passive Lysis 5x Buffer</td><td>Promega</td><td>E1941</td><td></td></tr><tr><td>GloMax 96 Microplate Luminometer</td><td>Promega</td><td>E6501</td><td></td></tr><tr><td>Sucrose</td><td>Nacalai tesque</td><td>30403-55</td><td></td></tr><tr><td>Luminometer plate</td><td>Greiner bio-one</td><td>655075</td><td></td></tr><tr><td>HepG2-NTCP1-myc-clone22</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV delta epsilon</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV/NL</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>50 ml tube</td><td>Violamo</td><td>1-3500-02</td><td></td></tr><tr><td>Anti-Myc antibody</td><td>Sigma-Aldrich</td><td>C3956</td><td></td></tr><tr><td>HBIG</td><td>Japan Blood Products Organization</td><td>-</td><td></td></tr><tr><td>IFN-&amp;beta;</td><td>Mochida Pharmaceutical</td><td>14987224005413</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr></tbody></table>

development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Figure 1 shows a schematic of the HBV genome, transcribed RNAs, virus proteins and their coding region on the genome. The reporter gene and its location in the genome are also indicated. Figure 2 shows the HBV reporter plasmid and helper plasmid containing the reporter gene. The pUC1.2HBV/NL reporter was constructed by deleting nucleotide positions 223-811 from the transcription initiation site of the preCore mRNA of pUC1.2HBV16 an...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Research

JoVE Journal

Immunology and Infection

13.2K Views.  National Center for Global Health and Medicine.  Hier beschrijven we een nieuw ontwikkelde hepatitis B-virus (HBV) reportersysteem de vroege stadia van de levenscyclus HBV controleren. Deze vereenvoudigde in vitro systeem zal helpen bij het screenen van anti-HBV middelen met een high-throughput strategie. 

Video: Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly ...

Biology

Suppressing Hepatitis B Virus Replication Using Viral Antigen-Specific T Cells

Source: Xiong, X., et al. <a href="https://app.jove.com/v/60043">Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice.</a> J. Vis. Exp. (2019).
The video demonstrates an assay using human induced pluripotent stem cell (iPSC)-derived CD8+ T cells to inhibit hepatitis B virus (HBV) replication in mice. These T cells, engineered with HBV-specific receptors, are transferred into transgenic mice. Replication-competent HBV genome plasmids are then injected directly into the liver, enabling viral DNA uptake. Once viral antigens are expressed and presented by hybrid MHC-I molecules, the engineered T cells recognize and bind to these complexes. This triggers the release of cytotoxic molecules, resulting in the destruction of HBV-infected liver cells and suppression of viral replication.

All procedures involving animal models have been reviewed by the local
institutional animal care committee and the JoVE veterinary review board. All ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Samenvatting

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