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>

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	<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. 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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. 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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&#956;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>&#8212;&#8212;</td>
			<td>Cell model for HBV infection</td>
		</tr>
		<tr>
			<td>293T-NE-3NRs</td>
			<td>Laboratory construction</td>
			<td>&#8212;&#8212;</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&#65288;FBS&#65289;</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>&#8212;&#8212;</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 ...

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Immunology and Infection

7.3K Views.  Shantou University Medical College. This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

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

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

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 ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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), ...

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

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

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

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

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

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

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

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

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 ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.