Name: detection of low copy number integrated viral dna formed by in vitro hepatitis b infection
Uploaded: 2018-11-07T15:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection at JoVE.com

This work received funding from the German Centre for Infection Research (DZIF), TTU Hepatitis Projects 5.807 and 5.704, the Deutsche Forschungsgemeinschaft (DFG) TRR179 (TP 15), and the Australian Centre for HIV and Hepatitis Virology Research.
We are indebted to Professor William Mason for his part in developing the original invPCR method and demonstrating it to us. We would like to thank Drs. Yi Ni and Florian A. Lempp for reagents (cell lines and HBV inoculum). We acknowledge Anja Rippert, Franziska Schlund, Sarah Engelhardt, and Dr. Katrin Sch&#246;neweis for technical assistance. We are grateful to Miriam Kleinig for proofreading, and to Professors Nicholas Shackel and Ralf Bartenschlager for continuous support.

1. Cell Infection and DNA Extraction
<ol>
	<li>Maintain cultured Huh7-NTCP cells8,9 in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% v/v fetal bovine serum, 1x Penicillin/Streptomycin, and 2 mM L-glutamine. 
	NOTE: This has previously been reported in detail40.</li>
	<li>Seed 2 x 105 cells/mL Huh7-NTCP cells into a 12-well plate with 1 mL of DMEM.</li>
	<li>If testing cell treatments (e.g., HBV inhibitors), apply them to the culture supernatant 4 h after seeding (1 day prior to HBV infection). For a negative control, apply 200 nM Myrcludex B (a potent HBV entry inhibitor41).</li>
	<li>Use heparin-column purified42 supernatant from HepAD3843 as an inoculum to infect cells at 1,000 VGE/cell in 500 &#181;L of culture media (DMEM supplemented with 10% v/v fetal bovine serum, 1x Penicillin/Streptomycin, 2 mM L-glutamine, and 2.5% v/v DMSO), containing 4% w/v polyethylene glycol 8000 [dissolved in 1x phosphate-buffered saline (PBS)].</li>
	<li>Culture the cells in a 37 &#176;C incubator (set at 5% CO2 and 90% humidity).</li>
	<li>Wash the cells twice with 1 mL of sterile 1x PBS at 16&#8211;24 h post-infection.</li>
	<li>Replace the culture media every 2 days following HBV infection until harvest.</li>
	<li>At day 3 post-infection, treat the cells with 5 &#181;M Tenofovir disoproxil and 10 &#181;M Lamivudine to limit production of HBV replicative intermediates that are amplifiable by invPCR.</li>
	<li>At day 5 post-infection, trypsinize the cells with 200 &#181;L of Trypsin-EDTA and resuspend them in 2 mL of DMEM (supplemented with 10% v/v fetal bovine serum, 1x Penicillin/Streptomycin, and 2 mM L-glutamine), also containing 5 &#181;M Tenofovir disoproxil and 10 &#181;M Lamivudine.</li>
	<li>Transfer the cell suspensions to a 6-well plate to induce one round of mitosis (which has been reported to induce loss of HBV cccDNA44 and other HBV DNA intermediates that are amplifiable by invPCR).</li>
	<li>At day 7 post-infection, trypsinize the expanded cells in 400 &#181;L of Trypsin-EDTA and resuspend the mixture in 1 mL of DMEM. Place the suspension in a 1.5 mL tube, pellet the cells by centrifugation at 500 x g for 5 min, and remove the supernatant by aspiration.</li>
	<li>(Optional) Store the cell pellets at -20 &#176;C until they are ready for DNA extraction.</li>
	<li>Extract the DNA from the cell pellet using a DNA extraction kit, as per the manufacturer&#8217;s instructions. Estimate the final DNA concentration using optical densitometry. 
	NOTE: The DNA yield in a 100 &#956;L elution volume is generally ~250-400 ng/&#956;L.</li>
</ol>
2. Inversion of DNA
<ol>
	<li>Aliquot ~1.5&#8211;2.5 &#956;g of the total DNA extract from step 1 into a 200 &#956;L PCR tube. Add the appropriate amount of restriction enzyme master-mix to result in a 40 &#956;L reaction volume containing 1x digest reaction buffer (e.g., CutSmart buffer) and 10 U NcoI-HF.</li>
	<li>Mix the reactions thoroughly and spin down in a small tube centrifuge. Incubate the restriction enzyme reaction in a PCR machine at 37 &#176;C for 1 h for optimal digestion efficiency.</li>
	<li>Inactivate the restriction enzyme by incubating at 80 &#176;C for 20 min.</li>
	<li>Transfer the entire restriction enzyme reaction to a 1.5 mL tube. Add 400 &#956;L of 1x T4 DNA ligase buffer and 500 U of T4 DNA ligase and mix it thoroughly. The large reaction volume encourages intra-molecular (as opposed to inter-molecular) ligation of the digested DNA fragments.</li>
	<li>Incubate the ligation reaction at room temperature for 2 h to ensure complete ligation.</li>
	<li>Inactivate the T4 DNA ligase at 70 &#176;C for 20 min.</li>
	<li>Add 10 &#956;L of 10% w/v sodium dodecyl sulfate to ensure complete inactivation of the T4 ligase.</li>
	<li>Mix the tubes by pulse vortexing and briefly spin down the reaction mix. Add NaCl to a final concentration of 100 mM and dextran (35&#8211;45 kDa) to a final concentration of 90 &#181;g/mL. Mix the tubes by pulse vortexing and briefly spin down the reaction mix.</li>
	<li>Add 900 &#956;L of 100% ethanol and mix by inversion. Precipitate the DNA at -20 &#176;C overnight.</li>
	<li>Pellet the precipitated DNA by centrifugation at 14,000 x g for 15 min. Remove the supernatant by aspiration with a P200 pipette. Wash the pellet with 500 &#956;L of 70% v/v ethanol and centrifugation at 14,000 x g for 15 min.</li>
	<li>Remove the ethanol by aspiration with a P200 pipette. Air-dry the DNA pellet at room temperature for 20 min.</li>
	<li>Dissolve the pellet in 20 &#956;L of H2O. Add 20 &#956;L of a restriction enzyme master-mix to result in a 40 &#956;L reaction volume containing 1x digest reaction buffer, 5 U of BsiHKAI, and 5 U of SphI-HF. Incubate the restriction enzyme reaction in a heat-block at 37 &#176;C (the optimal temperature for SphI-HF) for 1 h.</li>
	<li>Briefly spin down the reaction mix. Incubate the restriction enzyme reaction in a heat-block at 65 &#176;C (the optimal temperature for BsiHKAI) for 1 h.</li>
	<li>Briefly spin down the reaction mix.</li>
	<li>Store the inverted DNA at -20 &#176;C until the next step.</li>
</ol>
3. Nested PCR
<ol>
	<li>Remove potential amplicons from a reusable silicon mat seal using a DNA degradation solution, rinse the mat thoroughly with DNA-free water, and air-dry it at room temperature while preparing the PCR mixture.</li>
	<li>Prepare 1 mL of a 1x PCR mix containing the outer forward (5&#8217;-TTC GCT TCA CCT CTG CAC G-3&#8217;) and reverse (5&#8217;-AAA GGA CGT CCC GCG CAG-3&#8217;) primers at a concentration of 0.5 &#181;M.</li>
	<li>Add 170 &#956;L of the 1x PCR mix to wells A1 and E1 in a 96-well PCR plate.</li>
	<li>Add 120 &#956;L of the 1x PCR mix to wells B1 to H1.</li>
	<li>Add 10 &#956;L of the inverted DNA from step 2 to wells A1 and E1 (2 different inverted samples can be analyzed on the same PCR plate). Mix the reaction in each well by gently pipetting each about 10 times using a P1000 set to 100 &#956;L.</li>
	<li>Serially dilute samples from wells A1 to D1 at a ratio of 1:3 by transferring 60 &#956;L at each step. Mix the wells at each step by gently pipetting them about 10 times using a P1000 set to 100 &#956;L. Avoid forming bubbles. Repeat step 3.6 for well E1, diluting down to well H1.</li>
	<li>Aliquot 10 &#956;L of the reaction mixture using a multi-channel pipette from wells A1-H1 into wells A2-H2, A3-H3, and so on until reaching wells A12-H12 of the 96-well plate. Cover the PCR plate with the dry silicon mat from step 3.1, pressing firmly.</li>
	<li>Place the plate in a PCR machine and run the following program: 10 min at 95 &#176;C; 35 cycles of 15 s at 95 &#176;C, 15 s at 54 &#176;C and 3 min at 72 &#176;C; 7 min at 72 &#176;C; then, hold the plate at room temperature.</li>
	<li>Heat the pins of a 96-pin replicator to red-hot with a Bunsen burner and then cool for at least 5 min at room temperature.</li>
	<li>Fill the wells of a second PCR plate with 10 &#956;L of 1x PCR mix (containing a gel load-ready buffer) and the inner forward (5&#8217;-CGC ATG GAG ACC ACC GTG A-3&#8217;) and reverse (5&#8217;-CAC AGC CTA GCA GCC ATG G-3&#8217;) at 0.5 &#181;M.</li>
	<li>Carefully remove the silicon mat from the PCR plate of the 96-well plate from the first-round PCR, and avoid cross-contamination between wells.</li>
	<li>Use the cooled replicator to transfer the PCR products of the 96-well plate from first-round PCR to the newly-aliquoted 96-well plate. Carry out the nested PCR using the same conditions as in step 3.8, except for changing the initial denaturation step at 95 &#176;C from 10 min to 2 min.</li>
</ol>
4. PCR Product Isolation and Gel Extraction
<ol>
	<li>Analyze the PCR products by gel electrophoresis using a 96-well with 1.3% w/v agarose gel. For a 100 mL agarose gel, run at 200 V for 10&#8211;15 min.</li>
	<li>Excise the DNA bands from the agarose gels using disposable drinking straws. For each PCR product, place the straw and agarose gel plug into a 1.5 mL tube and trim the straw to size with scissors. 
	NOTE: It is sufficient to isolate bands only from those dilutions in which single PCR products can be resolved.</li>
	<li>(Optional) Store the tubes at -20&#176;C for later extraction.</li>
	<li>Eject the agarose plugs into each tube by squeezing on the end of the straw fragment. Add 300 &#956;L of gel extraction buffer and 5 &#956;L of gel extraction glass bead slurry to each tube.</li>
	<li>Extract the PCR products as per the manufacturer&#8217;s instructions for the gel extraction kit (except using half-volumes for washing steps) and elute the DNA from the beads with 30 &#956;L of water.</li>
	<li>Submit the purified DNA for Sanger sequencing (as per the supplier&#8217;s instructions) with the Forward primer used in the second-round nested PCR.</li>
</ol>
5. Sequence Analysis
<ol>
	<li>Confirm virus-cell DNA junctions by performing nucleotide BLAST analysis (using default settings aligning to the entire nucleotide collection). 
	NOTE: Representative results are detailed in Figure 3 below.
	<ol>
		<li>If only partial alignment of the sequence is observed, trim the 5&#8217; HBV DNA sequence before re-running a BLAST analysis.</li>
	</ol>
	</li>
	<li>Apply stringent selection criteria to determine if a given sequence represents a true integration junction as follows:
	<ol>
		<li>Include only sequences containing &#62;20 bp of a cellular sequence for confident mapping to the cellular genome.</li>
		<li>Disregard any sequences that contain restriction sites of any enzymes used within 10 bp of the supposed virus-cell integration junction, as they likely represent in vitro ligation events rather than true integration junctions.</li>
		<li>Disregard any sequences with obvious dissimilar peak fluorescence levels on either side of the supposed integration junction in sequencing chromatographs, as these are likely artifacts generated by cross-contamination during the sequencing reaction or capillary chromatography.</li>
		<li>Define unique integration events as those with the exact HBV and cellular sequences at the virus-cell junction. 
		NOTE: Repetition of unique integration events can result from clonal expansion of cells containing those integrations or cross-contamination during the PCR (which can be tested using negative-control reactions, further detailed in the representative results below). Repeated integrations into specific cellular sequences are expected to display different HBV terminal sites for each integration event, as non-homologous end-joining pathways are used15.</li>
	</ol>
	</li>
	<li>Calculate the integration frequency by multiplying the dilution factor of inverted DNA templates by the number of virus-cell junctions detected at that dilution, followed by normalization to the amount of total DNA input into the inversion reaction. Generally, the integration frequency is on the order of 1:104 cells.</li>
</ol>

S.U. is a co-applicant and co-inventor of patents protecting Myrcludex B as an HBV/HDV entry inhibitor. T.T. declares no conflicts of interest.

Before performing the protocol, it is important to note that this invPCR assay is a highly sensitive technique, capable of amplifying single copies of DNA template. Therefore, limiting contamination from PCR products is of utmost importance. General strategies to limit PCR product contamination include the following. (i) Establish physically separate areas for different steps of the method. Each area should have separate lab coats, and gloves should be changed when moving between these areas. We have listed these areas below in order from most to least likely to be contaminated: PCR product extraction and sequencing reaction set-up area (post-PCR); PCR template addition and flamed-pin transfer area (we have used PCR hoods with a decontaminating UV lamp with good results); DNA extraction and inversion area (pre-PCR); and a &#34;DNA template-free&#34; area used solely for preparing stock and PCR solutions. (ii) Be aware of air flow in the lab as a potential driver of cross-contamination. In particular, cross-contamination of PCR reactions during the flamed pin transfer step is most likely to occur and will lead to inaccurate quantification of integration frequency. PCR hoods can be used to limit these cross-currents. Negative control wells (e.g., Myrcludex B-treated samples or no template controls) in the PCR can be also used to test for cross-contamination. (iii) Limit potential PCR contaminants on pipettes and work surfaces by regularly wiping them down with a DNA degradation solution.
The protocol specified here is arranged to detect integration of a known infectious HBV clone generated from the HepAD38 cell line42. If the inoculum used is of a different HBV DNA sequence (e.g., from patient serum), then the HBV genome should be first sequenced to confirm compatibility of PCR primers and inversion design. In previously published studies19, we have used 3 sets of primers that bind conserved HBV DNA sequences flanking the expected site of HBV DNA integration junctions. Other primer sequences and protocols have been described to determine the genomic sequence of HBV44,45,46,47,48&#160;and may work successfully.
Furthermore, different HBV-susceptible cells (e.g., primary human hepatocytes, differentiated HepaRG, HepG2-NTCP cells) can be used; however, we have found that Huh7-NTCP cells provide the greatest signal-to-noise ratio, when considering the number of virus-cell DNA junctions that are amplified compared to the virus intermediate DNA that is amplified13. In particular, terminally differentiated cells such as primary human hepatocytes or differentiated HepaRG do not efficiently undergo mitosis, resulting in a high level of amplifiable HBV DNA replicative intermediates remaining within the cells. We have found that ~90% of amplified sequences represent HBV DNA rearrangements (and not integration events) in terminally-differentiated cells, compared to 70&#8211;50% in hepatoma cell lines13. These products are generally HBV DNA genomes containing large deletions in the surface and core open reading frames, or they can represent HBV quasi-species with an additional NcoI site prior to the SphI site. The amplification of these HBV species (including a schematic diagram) have been described in detail previously13.
There are several drawbacks to this method. Due to the laborious and multi-day nature of this protocol, invPCR is not suited to high-throughput analyses of a large number of samples. Furthermore, as it relies on limiting dilution titration, our method is not highly precise in quantification; although, it should easily measure log-level changes in integration frequency.
Moreover, our inversion protocol is only suited to detect integrations occurring between the DR2 and DR1 region of the HBV genome, as the majority of HBV integrations occur within this region49. NGS analysis of HBV patient tissues has shown that a large minority (up to ~50%) may also occur outside of this region49. New invPCR designs are theoretically able to detect these other integration sites, though they have not (to our knowledge) been carried out yet. Relatedly, due to the necessary restriction enzyme sites required downstream from the virus-cell junction sequence for the inversion reaction, invPCR does not detect all integrations occurring within the DR2 and DR1 regions of the HBV genome (i.e., we have estimated that ~10% of all integrations are detectable using in silico simulations13). However, when applied to a focused batch of samples with small numbers of different treatments, invPCR is one of the only practical methods for detecting integrated HBV DNA at a single base-pair resolution.
We therefore envision applications of this method serving a key role in finding viral (via mutations of the HBV inoculum), cellular (through knockout or overexpression of specific cellular genes, or through application of various drugs), and environmental (e.g., exposure to oxidative stress) factors that induce HBV DNA integration. With this method and newly developed HBV infection systems, we enable unprecedented control of these factors so that they can be well-isolated and better characterized. We also expect that this will lead to a key understanding of the cellular consequences of HBV DNA integration, including to what extent viral antigens (e.g., HBx or HBsAg50) are expressed from the integrated form, what controls this expression, and whether or not HBV integration significantly changes the cellular phenotype towards a more pro-oncogenic state. The results of these future studies will have a profound impact on the therapeutic strategies used to treat chronic hepatitis B and on the basic understanding of the virus itself.

HBV is a double-stranded DNA virus that can cause life-long chronic infection, leading to liver cirrhosis and hepatocellular carcinoma (HCC)1,2,3. While there are multiple molecular mechanisms driving HBV persistence4 (e.g., high stability of the epigenetic viral transcriptional template, evasion of immune surveillance, and low turnover of hepatocytes in the liver) and its associated risk of HCC initiation5,6 (e.g., chronic inflammation and activation of cellular stress pathways), HBV DNA integration into the host cell genome (a reported mechanism for both of these phenomena) has been poorly studied. A major reason is the lack of suitable in vitro infection systems for HBV that allow reliable detection of integration events. Here, we describe a recently-developed protocol for both in vitro generation and detection of HBV DNA integrations that can be used to elucidate the underlying molecular mechanisms and consequences thereof.
HBV replication and the formation of HBV DNA integration has been previously reviewed in detail7. Briefly, HBV enters hepatocytes using the Sodium Taurocholate Co-transporting Peptide (NTCP) as the main cellular receptor for infection8,9. The HBV nucleocapsids containing the HBV-relaxed circular DNA (rcDNA) genome enters the nucleus, where the rcDNA is converted into episomal covalently closed circular DNA (cccDNA). The nuclear cccDNA acts as the transcriptional template for viral mRNAs and pre-genomic RNA (pgRNA)10. HBV polymerase and pgRNA are then packaged into newly-formed nucleocapsids (composed of HBV core protein dimers). HBV pgRNA is reverse-transcribed within the nucleocapsid, resulting in either an rcDNA genome or a double-stranded linear DNA (dslDNA) genome11,12. These mature nucleocapsids containing HBV DNA genomes are finally enveloped and exported as virions.
Infection of hepatocytes by enveloped particles containing dslDNA molecules can result in viral integration into the host cell genome13, leading to replication-incompetent forms of HBV DNA7,14,15. HBV DNA integration occurs at the site of chromosomal double-stranded DNA breaks15. Accumulating evidence suggests that each integration event occurs in an essentially random position within the host cell genome16,17. In addition, HBV DNA integration occurs somewhat rarely, at a rate of 1 per 104 cells13,18,19,20. Important questions regarding HBV DNA integration remain unanswered, particularly regarding the exact molecular pathways involved, the dependence on viral and host factors, the viral antigens expressed from integrated forms, and their possible contribution to viral persistence7. We have set up an in vitro model to shed light on some of these questions.
The rarity of HBV DNA integration events (both with respect to integration rate per cell and to the copy number of each unique integration) in in vitro HBV infection models make them challenging to detect. Cell mitosis is limited in our in vitro system, as dividing cells do not support efficient infection. Thus, unlike in patient liver tissues where significant clonal expansion of hepatocytes occurs18,19,20, very few (1 to 2) copies of each integration are present in a given pool of cells infected in vitro. We have also found that integration mainly occurs during the initial infection of hepatocytes (and not continuously in chronically-infected hepatocytes)13. Accordingly, HBV DNA integration cannot be increased by simply culturing cells for a longer period.
In general, methods previously used to detect integrated HBV DNA, including Southern blot hybridization21,22,23,24,25, Alu-PCR26,27, cassette-mediated ligation PCR28, and whole genome sequencing29,30,31,32,33, do not have the sensitivity to detect single-copies of integrations. We and other researchers have used inverse nested PCR (invPCR) to detect hepadnaviral DNA integration in duck, woodchuck, and human infected livers13,14,18,19,34,35,36. Others have introduced procedural modifications to the invPCR technique that may alter the generation of false-positive signals and restrict the ability for quantification37. If the assay is carried out as described in this protocol, invPCR represents a specific and sensitive assay that identifies and quantifies (in absolute copy numbers) multiple HBV DNA integrations. The HBV-cell DNA junction is sequenced at a single-base pair resolution, allowing bioinformatical studies of viral and host DNA sequences at the sites of integration13.
We have previously described38&#160;results from invPCR on DNA of HBV-infected tissue extracted from a large range of sources, including snap-frozen liver tissues, paraffin-embedded liver sections, and extremely small tissue samples isolated by laser-microdissection19. This protocol outlines an updated version of an invPCR assay using DNA extracted from cell culture-derived tissues after in vitro infection, in which low copy numbers of integrations (1&#8211;2 copies per integration) are generated. The HBV DNA integrations formed in vitro resemble those found in patient tissues with respect to their distribution over the cellular genome and junction within the viral sequence that is integrated13,16, suggesting a comparable pathway to within an infected liver.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s PBS</td><td>PAA</td><td>H15-002</td><td></td></tr><tr><td>DMEM medium</td><td>Thermo Fisher Scientific</td><td>41965</td><td></td></tr><tr><td>Fetal bovine serum</td><td>PAA</td><td>A15-151</td><td>Heat-inactivate before use</td></tr><tr><td>Penicillin, 10,000 U/mL; Streptomycin 10mg/ml, 100&amp;times;</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>L-glutamine, 200 mM</td><td>PAA</td><td>M11-004</td><td></td></tr><tr><td>Trypsin, 0.5 mg/mL; EDTA, 0.22 mg/mL, 1&amp;times;</td><td>PAA</td><td>L11-004</td><td></td></tr><tr><td>PEG, MW 8000</td><td>Sigma-Aldrich</td><td>89510</td><td>Stock at 40% w/v in 1xPBS, autoclave before use</td></tr><tr><td>DMSO for spectroscopy</td><td>Merck</td><td>102950</td><td></td></tr><tr><td>Tenofovir disoproxil</td><td>Sigma-Aldrich</td><td>CDS021622</td><td>Dissolve in DMSO</td></tr><tr><td>Lamivudine</td><td>Sigma-Aldrich</td><td>L1295</td><td>Dissolve in DMSO</td></tr><tr><td>NucleoSpin Tissue kit</td><td>Macherey Nagel</td><td>740952.250</td><td></td></tr><tr><td>NanoDrop 2000/2000c Spectrolphotometer</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>&lt;em&gt;Nco&lt;/em&gt;I-HF</td><td>NEB</td><td>R3193L</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202T</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>433209</td><td></td></tr><tr><td>Dextran (35-45 kDa)</td><td>Sigma-Aldrich</td><td>D1662-10G</td><td></td></tr><tr><td>Absolute Ethanol</td><td>Sigma-Aldrich</td><td>32205-1L-D</td><td></td></tr><tr><td>&lt;em&gt;Bsi&lt;/em&gt;HKAI</td><td>NEB</td><td>R0570L</td><td></td></tr><tr><td>&lt;em&gt;Sph&lt;/em&gt;I-HF</td><td>NEB</td><td>R3182L</td><td></td></tr><tr><td>Amplitaq Gold Taq kit</td><td>Thermo Fisher Scientific</td><td>4331816</td><td>Use for first nest PCR</td></tr><tr><td>Silicon sealing Mats for 96-Well PCR Plates</td><td>Biorad</td><td>2239442</td><td></td></tr><tr><td>DNAZap PCR DNA Degradation Solution</td><td>Thermo Fisher Scientific</td><td>AM9890</td><td></td></tr><tr><td>96-well PCR plates</td><td>Sigma Aldrich</td><td>CLS6509 SIGMA</td><td></td></tr><tr><td>96-pin replicator</td><td>Thermo Fisher Scientific</td><td>250520</td><td></td></tr><tr><td>GoTaq Flexi PCR kit</td><td>Promega</td><td>M8295</td><td>Use for second nest PCR, use green buffer for easy loading of agarose gel</td></tr><tr><td>Biozym LE Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>QIAEX II Gel extraction kit</td><td>QIAGEN</td><td>20051</td><td></td></tr></tbody></table>

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.

A schematic representation of the invPCR technique is shown in Figure 1. An example agarose gel electrophoresis of a successful invPCR is shown in Figure 2 before and after the PCR product isolation. Figure 3 shows the BLAST analysis output from step 5.1 in the cases of (i) amplification of virus-cell DNA junction and (ii) amplification of an HBV DNA replicative intermediate.
Expected results from positive controls: Huh7-NTCP cells infected with heparin-purified HBV stocks as described above (Figure 1) can serve as a positive control. In this instance, single PCR products should be achieved by the second or third 1:3 dilution of each sample (corresponding to ~104 cell equivalents per dilution, Figure 2). At these dilutions, approximately 50% of products will represent true virus-cell DNA junctions, while the other half represents amplification of HBV DNA intermediates (Figure 3).
In previous studies13, we have found few instances of repeated virus-cell junctions, suggesting no cellular genomic sites of preferential HBV DNA integration. We also find that &#62;80% of virus-cell junctions occur between nucleotides 1732 and 1832 (according to the nucleotide numbering of HBV genotype D, GenBank Accession #U95551.1), where the latter represents the right-hand terminus of the HBV dslDNA form. Sequences of true virus-cell junctions found through our method in in vitro experiments are publicly available on GenBank (Accession numbers MH057851 to MH058006).
Expected results from negative controls: Either uninfected Huh7-NTCP cells or inoculated Huh7-NTCP cells pre-treated with Myrcludex B can act as negative controls. InvPCR analysis of DNA extracted from these cells should produce no PCR products. Running these negative-control samples on the same PCR plate as positive samples allows for testing of cross-contamination during the nested-PCR process. The most stringent test for cross-contamination is the running of a negative-control sample in wells A-D and the positive sample in wells E-H on a 96-well plate. In this case, the most concentrated dilution of the positive sample is run in a row directly adjacent to the least concentrated dilution of the negative sample. If amplification products are observed in negative-control samples, institute the measures suggested in the discussion to minimize cross-contamination events.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58202/58202fig1.jpg" /> 
Figure 1: Schematic figure of the invPCR process to detect HBV DNA integrations. After HBV infection, only a minority of Huh7-NTCP cells contain HBV dslDNA (red) integrated into the host cell genome (red), depicted in the top section. Total DNA is then extracted from the cells (step 1), inverted (step 2), and amplified by nested PCR (step 3). Shown are the relative positions of the restriction enzyme sites (NcoI and BsiHKAI) in the excised virus-cell DNA junction. The figure is adapted from a previous publication13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58202/58202fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58202/58202fig2.jpg" /> 
Figure 2: Agarose gel electrophoresis and subsequent gel extraction of a successful invPCR. &#34;M&#34; represents DNA marker ladder. Each row of 12 represents technical replicates at a single dilution on the PCR plate. Two inverted DNA samples can be run per PCR plate/agarose gel. The PCR products for each dilution should titrate out in a ~1:3 ratio. Extraction of the products from the two least concentrated dilutions where single bands can be easily isolated is generally sufficient, as HBV DNA integration rate is determined by end-point titration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58202/58202fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58202/58202fig3.jpg" /> 
Figure 3: BLAST analysis of invPCR product sequences. As long tracts of sequence are generated from Sanger sequencing, the source of DNA sequences is generally unambiguous. Strong alignment to HBV and cellular genomes is observed in different areas of the FASTA sequence file generated from Sanger sequencing in the top panel, suggesting a true virus-cell DNA junction. In the bottom panel, the sequence aligns only to fragments of the HBV genome, suggesting a product generated by the amplification of a rearranged HBV DNA genome. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58202/58202fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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 ...

detection-low-copy-number-integrated-viral-dna-formed-vitro-hepatitis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.3K Views.  Heidelberg University Hospital. Deze methode kan worden gebruikt om belangrijke vragen in hepatitis B virologie te beantwoorden, zoals het vinden van cellulaire of virologische factoren die de HPV-DNA-integratie beïnvloeden. Er zijn meerdere voordelen aan deze techniek. Het is relatief goedkoop, en gemakkelijk uit te voeren, analyse van de resultaten is eenvoudig, en het is zeer gevoelig, tot enkele kopieën.Hoewel deze methode kan worden gebruikt om inzicht te geven in HBV-integratie, kan deze ook worden gebruikt v...

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

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

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 ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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

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.

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), ...

"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 ...

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

An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection

Plate HepG2 cells, stably expressing sodium taurocholate, co-transporting polypeptide, or NTCP that are susceptible to HBV infection, and incubate at 37 degrees Celsius and 5% CO2.
One day before infection, plate approximately 5 x 104 HepG2/NTCP cells into the wells of a 96-well collagen-coated plate in 0.1 milliliters of culture medium. Thaw recombinant HPV in a 37 degrees Celsius water bath, until there is a small bit of ice remaining in the vial.
Prepare medium for infection by combining 78 microliters of fresh culture medium, two microliters of DMSO, 10 microliters of 40% PEG8000 in 1X PBS, and 10 microliters of recombinant HPV. Add 100 microliters of recombinant HBV solution to each well of a 96-well plate.
One day after infection, use 300 microliters of PBS to wash the cell three times, to remove the contaminating reporter protein from the virus fraction. Add 200 microliters of culture medium containing 2% DMSO to the infected cells, and incubate for one week or less.
To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 microliters of lysis buffer to the infected cells. Rock the culture plate for five minutes, then, centrifuge at 2000 times g for five minutes. Add 50 microliters of the reporter substrate to a luminometer plate, then, add 20 microliters of the cell lysate to the plate, and mix by briefly shaking. Finally, place the plate in the luminometer and initiate reading.

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

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