This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.), Research Fund for International Young Scientists (No. 81950410640 to W.I.); The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

Culture, collection of supernatants from HepG2.2.15 cells and HBV infection should be performed in biosafety level II (P2) or biosafety level III (P3) laboratory according to the biosafety guidance in the country. Laboratory safety practices should be followed to ensure the safety of laboratory personnel, and all should be vaccinated and detected HBsAb positive before performing HBV experiments. Observe the state of the cells at every step before proceeding to the next step. Human serum samples were used in accordance with the approval obtained by the Institutional Review Board of Shantou University Medical College.
1. HepG2.2.15 cell culture
NOTE: HepG2.2.15 cell supernatant, HepG2.2.15 cell supernatant concentrate and all the tips, flasks, plates, and tubes that come in contact with HepG2.2.15 should be disposed after being soaked in 2% virucide overnight.
<ol>
	<li>Remove the vial containing HepG2.2.15 cells from liquid nitrogen and thaw by gently swirling the vial in a 37 &#176;C water bath. 
	NOTE: To reduce the possibility of contamination, keep the cap out of the water. Thawing should be rapid (approximately 2 min).</li>
	<li>Remove the vial from the water bath as soon as the cells are thawed and disinfect it with 70% ethanol. 
	NOTE: From this point perform all steps under strict aseptic conditions.</li>
	<li>Transfer the cells to a 25 cm2 tissue culture flask and add 4 mL of complete culture medium (DMEM containing 10% FBS, penicillin 100 U/mL, streptomycin 0.1 mg/mL). Incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2.</li>
</ol>
2. Collection of HepG2.2.15 cell culture supernatant
<ol>
	<li>Culture HepG2.2.15 cell in T25 flasks at 37 &#176;C, 5% CO2 in a humidified incubator. Change the medium every 3 days.</li>
	<li>Collect the cell supernatant in a 50 mL centrifuge tube. Close the lid firmly and wrap it with a paraffin film.</li>
	<li>Store the HepG2.2.15 supernatant at -20 &#176;C to keep HBV virus active.</li>
</ol>
3. Concentrating HepG2.2.15 supernatant
<ol>
	<li>Remove HepG2.2.15 supernatant from -20 &#176;C and thaw at 4 &#176;C.</li>
	<li>To remove the cell fragments, filter the HepG2.2.15 supernatant with a 0.45 &#181;m membrane to a new 50 mL centrifuge tube. First, block the filter by filtering 10 mL of DMEM with 10% FBS before filtering the virus, then filter the cell culture supernatant.</li>
	<li>Add 14 mL of filtered supernatant to a virus concentrator column and close the lid. Centrifuge the column at 3,200 x g for 35 min in a horizontal spinning centrifuge. Collect the HepG2.2.15 supernatant concentrate (about 200 &#181;L) into a 1.5 mL tube.</li>
	<li>Wash the column with DMEM once by adding 200 &#181;L of DMEM and collect it into a 1.5 mL tube.</li>
</ol>
4. Detection of HBV DNA in supernatant concentrate
<ol>
	<li>Turn on the dry block heater and set the temperature to 100 &#176;C.</li>
	<li>To ensure that the concentration of HBV DNA in the HepG2.2.15 supernatant concentrate is within the standard curve range, dilute the concentrate 20-fold by adding 10 &#181;L of HepG2.2.15 supernatant concentrate to 190 &#181;L of DMEM in a 1.5 mL microcentrifuge tube. Add 200 &#181;L of HepG2.2.15 supernatant, negative control (HBV-negative serum from healthy donor), HBV positive control (HBV-positive serum from HBV patient) and four dilutions of the quantitative references provided in the kit (2.0 x 106, 2.0 x 105, 2.0 x 104, 2.0 x 103 GEq /mL), respectively to separate 1.5 mL microcentrifuge tubes.</li>
	<li>Add 450 &#181;L of HBV DNA extraction buffer from the kit to all the above eight 1.5 mL tubes, vortex for 15 s and spin down for 10 s. Incubate at 100 &#176;C for 10 min in dry block heater. Centrifuge at 12, 000 x g for 5 min at room temperature.</li>
	<li>Add 27 &#181;L of PCR master mix and 3 &#181;L of Taq polymerase enzyme to the PCR tubes placed on ice. 
	NOTE: PCR master mix provided in the kit contain the primers against the conserved region of HBV DNA.</li>
	<li>Add 20 &#181;L of centrifuged supernatant liquid to the PCR tubes placed on ice. Cover tightly and spin down for 10 s.</li>
	<li>Use the following conditions on a real-time PCR machine: 93 &#176;C for 2 min, 10 cycles of 93 &#176;C for 45 s followed by 55 &#176;C for 60 s; then 30 cycles of 93 &#176;C for 30 s followed by 55 &#176;C for 45 s to carry out real-time PCR. 
	NOTE: The master mix provided in the commercial kit has primers against the conserved region of HBV DNA.</li>
	<li>Export the Ct values obtained and generate a standard curve as shown in Table 1 and Figure 2.</li>
	<li>Based on the standard curve, calculate the HBV DNA concentration of HepG2.2.15 supernatant concentrate diluted 20x as shown in Table 2. Calculate the HBV DNA concentration of HepG2.2.15 supernatant concentrate (Table 2).</li>
	<li>Divide the HepG2.2.15 supernatant concentrate in aliquots and store at -80 &#176;C until use.</li>
</ol>
5. In vitro infection of HepG2-NE and 293T-NE-3NRs cells
<ol>
	<li>Prepare the following infection medium in DMEM/F-12: 10% FBS, 4 mM glutamine supplement, 0.1 mM NEAA, 1 mM Sodium pyruvate, 100 U/mL penicillin, 100 &#956;g/mL streptomycin, 1 &#956;g/mL puromycin, 40 ng/mL dexamethasone, 20 &#956;g/mL hydrocortisone and 5 &#956;g/mL insulin. Store at 4 &#176;C. 
	NOTE: These NTCP positive cells are puromycin-resistant9.</li>
	<li>Add 0.1% gelatin to 5 wells of 48 well-plate, 200 &#956;L to each well. Incubate the plate at 37 &#176;C for 2 h, discard the supernatant. Incubate the plate at 37 &#176;C for another 2 h.</li>
	<li>Seed HepG2-NE at a density of 1 x 104 cells in 2 wells each. Seed 293T-NE-3NRs cells at a density of 1 x 104 cells in 2 wells each (all-trans retinoic acid at 1 &#956;mol/L and clofibric acid at 1 mmol/L final concentrations were added to the medium to activate the&#160;RXR&#945; and&#160;PPAR&#945; nuclear hormone receptors, respectively). Seed 293T-NE in 1 well at a density of 1 x 104 cells. 
	NOTE: Passage the cells at sub-confluence using 0.25% trypsin. Count the cell number and then seed the cells to an appropriate density.</li>
	<li>Incubate the cell at 37 &#176;C, 5% CO2 in a humidified incubator for 24 h. Observe the cells after 24 h and proceed with infection if the cells are healthy.</li>
	<li>Prepare the infection complex as mentioned in Table 3, add HBV (HepG2.2.15 supernatant concentrate), DMSO, PEG8000 and infection medium to 1.5 mL microcentrifuge tube. Prepare CsA stock by dissolving it in DMSO to a concentration of 5 mM and keep the stock at -20 &#186;C. The working solution of CsA is 5 &#956;M. Seed 1&#215;104 cells per well of the 48-well plate. Add 100 &#956;L of HepG2.2.15 supernatant concentrate (containing HBV 1.5063 &#215;107 GEq/mL) to infect cells at 150 GEq/cell.</li>
	<li>Add 500 &#956;L of the infection complex to each well and incubate the cells at 37 &#176;C, 5% CO2 in humidified incubator for 24 h.</li>
	<li>Wash the cells twice with 500 &#956;L of PBS before changing the medium. Add 1 mL of the medium in each well. If the cell state is poor and some cells are floating, change the medium directly. This is day 1 of infection. Change the medium gently every 2 days.</li>
	<li>On day 11, wash the cells twice with 500 &#956;L of PBS and fix the cells with ice cold methanol for immunofluorescence.</li>
</ol>
6. HepG2.2.15 co-culture with HepG2-NE / 293T-NE-3NRs / 293T-NE
<ol>
	<li>Add 1 mL/well of 0.1% gelatin to 5 wells of 6 well-plate. Incubate the plate at 37 &#176;C for 2 h and discard the supernatant. Incubate the plate again for another 2 h at 37 &#176;C to completely dry the well.</li>
	<li>Seed 1 x 105 cells/well of HepG-NE into 2 wells, 293T-NE-3NRs into 2 wells, and 293T-NE into 1 well of the plate. Seed 1 x 105 cells/well of HepG2.2.15 onto five membrane inserts (24 mm, 0.4 &#956;m pore diameter, uncoated) in a separate 6 well plate. Incubate the cells at 37 &#176;C, 5% CO2 in a humidified incubator for 24 h.</li>
	<li>After 24 h, if the cells are all adherent and in good state, prepare for co-culture. Discard the medium in the 6-well-plate and cell membrane inserts. Place the membrane inserts with HepG2.2.15 into the 6 well plate seeded with HepG-NE, 293T-NE-3NRs and 293T-NE by tweezers. Incubate the cell at 37 &#176;C, 5% CO2 in a humidified incubator, change the medium every 3-4 days. 
	NOTE: Set groups as following: Well 1: 293T-NE co-culture with HepG 2.2.15; Well 2: HepG2-NE co-culture with HepG 2.2.15; Well 3: 293T-NE-3NRs co-culture with HepG2.2.15; Well 4: HepG2-NE co-culture with HepG 2.2.15 (add CsA); Well 5: 293T-NE-3NRs co-culture with HepG2.2.15 (add CsA); Add the complete medium to well number 1, 2 and 3, add the medium with 5 &#181;M CsA to well number 4 and 5 well, about 9 mL for each well, make sure the media are in contact in order for virus particles to migrate from transwells to infect cells.</li>
	<li>After 10 days, remove the membrane inserts, add PBS to the 6-well-plate and wash gently twice, fix the cells with ice cold methanol for immunofluorescence.</li>
</ol>
7. Perform immunofluorescence for HBcAg
<ol>
	<li>Fix the cells with ice cold methanol at -20 &#176;C for more than 3 h. Discard the methanol. Wash the cells with PBS for 10 min at room temperature on shaker, repeat for 3 times.</li>
	<li>Add 5% BSA (100 &#956;L/48-well-plate, 500 &#956;L/6-well-plate), shake on horizontal shaker for 1 h at 40 rpm and room temperature.</li>
	<li>Add the HBcAg primary antibody solution (primary antibody: 5% BSA solution =1:200, 100 &#956;L / 48-well-plate, 500 &#956;L / 6-well-plate), shake overnight at 4 &#176;C.</li>
	<li>Wash wells with PBST (PBS with 0.1% tween 20), for 10 min at room temperature on shaker, repeat for 3 times.</li>
	<li>Add the second antibody solution (594 labeled antibody raised in goat against rabbit IgG: PBS = 1:1,000, 100 &#956;L /48-well-plate, 500 &#956;L / 6-well-plate), cover the plate with foil and shake for 2 h at room temperature.</li>
	<li>Wash again with PBST for three times, shake for 10 min each.</li>
	<li>Add 1 &#956;g/mL DAPI, shake for 2 min. Wash twice with PBST on a shaker for 5 min each. Discard PBST. Add PBS and observe under immunofluorescence microscope.</li>
</ol>

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+hepatitis+report,+2017.">Global hepatitis report, 2017.</a> World Health Organization. , (2017).</li><li>Yoffe, B., Burns, D. K., Bhatt, H. S., Combes, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrahepatic+hepatitis+B+virus+DNA+sequences+in+patients+with+acute+hepatitis+B+infection.">Extrahepatic hepatitis B virus DNA sequences in patients with acute hepatitis B infection.</a> Hepatology. 12, 187-192 (1990).</li><li>Mason, A., Wick, M., White, H., Perrillo, R. <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+replication+in+diverse+cell+types+during+chronic+hepatitis+B+virus+infection.">Hepatitis B virus replication in diverse cell types during chronic hepatitis B virus infection.</a> Hepatology. 18, 781-789 (1993).</li><li>Yan, 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=Sodium+taurocholate+cotransporting+polypeptide+is+a+functional+receptor+for+human+hepatitis+B+and+D+virus.">Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.</a> eLife. 1, 00049 (2012).</li><li>Tang, H., McLachlan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+hepatitis+B+virus+by+nuclear+hormone+receptors+is+a+critical+determinant+of+viral+tropism.">Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 1841-1846 (2001).</li><li>Sells, M. A., Chen, M. L., Acs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hepatitis+B+virus+particles+in+Hep+G2+cells+transfected+with+cloned+hepatitis+B+virus+DNA.">Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 1005-1009 (1987).</li><li>Sells, M. A., Zelent, A. Z., Shvartsman, M., Acs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+intermediates+of+hepatitis+B+virus+in+HepG2+cells+that+produce+infectious+virions.">Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.</a> Journal of Virology. 62, 2836-2844 (1988).</li><li>Watashi, 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=Cyclosporin+A+and+its+analogs+inhibit+hepatitis+B+virus+entry+into+cultured+hepatocytes+through+targeting+a+membrane+transporter,+sodium+taurocholate+cotransporting+polypeptide+(NTCP).">Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).</a> Hepatology. 59, 1726-1737 (2014).</li><li>Yang, X., 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=Defined+host+factors+support+HBV+infection+in+non-hepatic+293T+cells.">Defined host factors support HBV infection in non-hepatic 293T cells.</a> Journal of Cell and Molecular Medicine. 24, 2507-2518 (2020).</li><li>Xia, Y., 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=Human+stem+cell-derived+hepatocytes+as+a+model+for+hepatitis+B+virus+infection,+spreading+and+virus-host+interactions.">Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions.</a> Journal of Hepatology. 66, 494-503 (2017).</li><li>Ortega-Prieto, A. M., Cherry, C., Gunn, H., Dorner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Model+Systems+for+Hepatitis+B+Virus+Research.">In Vivo Model Systems for Hepatitis B Virus Research.</a> ACS Infectious Diseases. 5, 688-702 (2019).</li><li>Liang, T. J. <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:+the+virus+and+disease.">Hepatitis B: the virus and disease.</a> Hepatology. 49, 13-21 (2009).</li><li>Nie, Y. Z., 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=Recapitulation+of+hepatitis+B+virus-host+interactions+in+liver+organoids+from+human+induced+pluripotent+stem+cells.">Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells.</a> EBioMedicine. 35, 114-123 (2018).</li><li>Nishinakamura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+kidney+organoids:+progress+and+remaining+challenges.">Human kidney organoids: progress and remaining challenges.</a> Nature Reviews Nephrology. 15, 613-624 (2019).</li></ol>

The authors have nothing to disclose.

Here, we introduce protocols for HBV infection in non-hepatic 293T-NE-3NRs and hepatoma-based HepG2-NE cells. 293T-NE-3NRs were suitable for HBV infection at both high and low GEq. The following critical steps need to be taken into consideration while using our protocol. The cell status is an important factor for a successful infection. The infection medium must be changed timely after the initial period of HBV infection. 293T-NE-3NRs cells are typically fragile following infection with high viral titers. Therefore, thes...

Hepatitis B affects the lives of more than 2 billion people and is one of the major threats to public health. Approximately 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, causing a big burden to the society1. Hepatocytes are not the only cells infected by HBV and other cells in non-hepatic tissue, such as kidney and testis, are also infected by this virus2,3. Currently, HBV infection cell models are limited to human hepatocytes with only few non-hepatic cell line models. This hampers the study of HBV infection and HBV-related pathology of non-hepatic tissues. Here we present protocols to study HBV infection in non-hepatic 293T cells as well as in hepatoma-based cells.
Sodium taurocholate co-transporting polypeptide (NTCP) is a functional receptor for human hepatitis B and hepatitis D virus4. Hepatocyte nuclear factor 4&#945; (HNF4&#945;), retinoid X receptor &#945; (RXR&#945;) and peroxisome proliferator-activated receptor &#945; (PPAR&#945;) are liver-enriched transcription factors restricting viral tropism of HBV. They have been verified to promote HBV pregenomic RNA synthesis and support HBV replication in a nonhepatoma cell line5. We constructed three different cell lines, HepG2 cell lines expressing NTCP (HepG2-NE), 293T cell line expressing NTCP (293T-NE) and 293T cell line expressing four host genes; NTCP, HNF4&#945;, RXR&#945; and PPAR&#945; (293T-NE-3NRs). Two methods for HBV infection were developed based on 293T-NE-3NRs (Figure 1). The first method uses inoculation in 293T-NE-3NRs with high viral genome equivalence (high GEq (150), DMSO and PEG8000) for 24 h. The second method employs co-culturing 293T-NE-3NRs with HepG2.2.15, which can produce HBV particles (low GEq (about 1.83) without DMSO and PEG8000), thus closely emulating natural conditions.
HepG2.2.15 cells are derived from the HepG2 line and chronically secrete infectious HBV, as well as subviral particles into the culture medium6,7. Cyclosporin A (CsA) is an immunosuppressant clinically used for the suppression of xenograft rejection. Studies have shown that CsA inhibits HBV entry into cultured hepatocytes by inhibiting the transporter activity of NTCP and blocking the binding of NTCP to large envelope proteins8.
HepG2-NE cells were used as positive control whereas CsA treated cells were used as negative control. By comparing with the positive and negative control groups, we can find out which host genes play a critical role in HBV infection. Additionally, through this mode of HBV infection, we can also find other novel mechanisms or host genes involved in HBV infection.

<table><tbody><tr><td>0.45&amp;mu;m membrane filter</td><td>Millex-HV</td><td>SLHU033RB</td><td>Filter for HepG 2.2.15 supernatant</td></tr><tr><td>293T-NE</td><td>Laboratory construction</td><td>&amp;mdash;&amp;mdash;</td><td>Cell model for HBV infection</td></tr><tr><td>293T-NE-3NRs</td><td>Laboratory construction</td><td>&amp;mdash;&amp;mdash;</td><td>Cell model for HBV infection</td></tr><tr><td>594 labeled goat against rabbit IgG</td><td>ZSGB-BIO</td><td>ZF-0516</td><td>For immunofluorescence assay,second antibody</td></tr><tr><td>6-well plate</td><td>BIOFIL</td><td>TCP010006</td><td>For co-culture</td></tr><tr><td>Amicon Ultra 15 ml</td><td>Millipore</td><td>UFC910008</td><td>For concentration of HepG 2.2.15 supernatant</td></tr><tr><td>BSA</td><td>Beyotime</td><td>ST023</td><td>For immunofluorescence assay</td></tr><tr><td>Cyclosporin A</td><td>Sangon biotech</td><td>59865-13-3</td><td>inhibitor of HBV infection</td></tr><tr><td>DAPI</td><td>Beyotime</td><td>C1006</td><td>For nuclear staining</td></tr><tr><td>Diagnostic kit for Quantification of Hepatitis B Virus DNA(PCR-Fluorescence Probing)</td><td>DAAN GENE</td><td>7265-2013</td><td>For HBV DNA detection</td></tr><tr><td>DMEM</td><td>HyClong</td><td>SH30243.01</td><td>For culture medium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td>For improvement of infection efficiency</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>CLARK Bioscience</td><td>FB25015</td><td>For culture medium</td></tr><tr><td>Fluorescence microscope</td><td>ZEISS</td><td>Axio observer Z1</td><td>For immunofluorescence assay</td></tr><tr><td>HepG2-NE</td><td>Laboratory construction</td><td>&amp;mdash;&amp;mdash;</td><td>Cell model for HBV infection</td></tr><tr><td>HBcAg antibody</td><td>ZSGB-BIO</td><td>ZA-0121</td><td>For immunofluorescence assay, primary antibody</td></tr><tr><td>PBS</td><td>ZSGB-BIO</td><td>ZLI-9062</td><td>For cell wash</td></tr><tr><td>PEG8000</td><td>Merck</td><td>P8260</td><td>For infection medium</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Thermo Fisher</td><td>10378016</td><td>For culture medium</td></tr><tr><td>Transwell</td><td>CORNING</td><td>3412</td><td>For co-culture</td></tr><tr><td>Tween 20</td><td>sigma-Aldrich</td><td>WXBB7485V</td><td>For PBST</td></tr><tr><td>Virkon</td><td>Douban</td><td>6971728840012</td><td>Viruside</td></tr></tbody></table>

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;

We constructed pSIN-NTCP-EGFP plasmid expressing NTCP and EGFP fusion and with puromycin resistance. The plasmid was transfected into HepG2 and 293T cells to construct stable cell lines HepG2-NE and 293T-NE expressing NTCP and EGFP. Plasmids (pSIN-HNF4&#945;, pSIN-RXR&#945;, pLV-PPAR&#945;-puro-flag) with puromycin resistance and expression were transfected into 293T-NE cells to construct a stable cell line expressing 4 host genes9. The expression of NTCP-EGFP can be observed by green fluorescence...

Watch this Scientific Journal Video about Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells at JoVE.com

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

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 ...

modeling-hepatitis-b-virus-infection-in-non-hepatic-293t-ne-3nrs-cells

Research

JoVE Journal

Immunology and Infection

7.3K Views.  Shantou University Medical College. HBV virale replicatie in extrahepatic speelt een belangrijke rol in de pathogenese van extrahepatische syndromen. Momenteel zijn celkweekmodellen voor het starten van extrahepatische HBV-infectie beperkt. We presenteren een in vitro niet-leverinfectiemodel om nieuwe hostfactoren te identificeren die hbv-replicatie beïnvloeden en hbv-gerelateerde nierziekten te onderzoeken.Vergeleken met traditionele HBV-infectiemodellen hebben we 293T-cellijn, 293T-NE-3NR, aangenomen en gecoördineerd...

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

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

Cancer Research

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

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 ...

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Bioengineering

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 ...

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 ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video