an in vitro recombinant virus reporter system for the detection of viral infection

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

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

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

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nano-Glo Luciferase Assay Regent</td>
			<td>Promega</td>
			<td>N1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol (PEG) 6000</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol (PEG) 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr>
			<td>Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Nishitsuji, H., et al. Cancer. Sci. (2015)</td>
		</tr>
		<tr>
			<td>pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Nishitsuji, H., et al. Cancer. Sci. (2015)</td>
		</tr>
		<tr>
			<td>pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Nishitsuji, H., et al. Cancer. Sci. (2015)</td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr>
			<td>HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>1.49872E+13</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Nishitsuji, H., et al. <a href="https://app.jove.com/v/54849">Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle.</a> J. Vis. Exp. (2017).
This video demonstrates an in vitro technique to detect hepatitis B virus (HBV) infection via a recombinant HBV reporter system. Upon infecting host cells, the recombinant viral genome expresses the luciferase reporter enzyme, which produces bioluminescence upon oxidizing its substrate &#8212; helping detect viral infection.

1. Production of Recombinant HBV Encoding the Reporter Protein

	Preparation of HepG2 cells
	
		Prepare cell culture medium (Dulbecco&#39;s modified ...

Take a human liver cancer cell culture, genetically modified to express sodium taurocholate co-transporting polypeptide or NTCP &#8212; a transmembrane receptor.
Add recombinant hepatitis B virus, or HBV. The virus comprises an outer envelope containing surface proteins and an inner nucleocapsid enclosing the genome. Insertion of a luciferase reporter gene inhibits the virus&#39;s reproductive ability.
Viral envelope protein binds to the host cell&#39;s NTCP receptor.
NTCP interacts with the epidermal growth factor receptor, EGFR, and oligomerizes, inducing EGFR-mediated endocytosis of the virus.
The viral envelope fuses with endosome membrane to release the nucleocapsid, which disassembles at the nuclear pore complex.
The viral genome enters the nucleus and converts to covalently closed circular DNA or cccDNA &#8212; a stable template &#8212; encoding viral proteins, including luciferase.
At predefined intervals, harvest the cells and lyse to release intracellular luciferase.
Introduce a reporter substrate. Luciferase oxidizes the substrate, producing a signal.
A post-infection increase in luciferase activity indicates continuous expression of the viral genome.

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In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

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We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

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Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

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

Transkrypcja

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