Name: a cell culture model for producing high titer hepatitis e virus stocks
Uploaded: 2020-06-26T14:30:00
Description: Watch this Scientific Journal Video about A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks at JoVE.com

We are grateful to Suzanne Emerson for the hepatitis E virus p6 clone. HEV-specific rabbit hyperimmune serum was kindly provided by Rainer Ulrich, Friedrich Loeffler Institute, Germany. Moreover, we thank all members of the Department of Molecular and Medical Virology at the Ruhr University Bochum for their support and discussion. Figures 1-7 were generated with BioRender.com.

NOTE: All experiments are performed under BSL-2 condition. All materials that get in contact with Hepatitis E virus RNA or infectious virus must be rinsed properly with 4% Kohrsolin FF from a waste container inside the hood prior to disposal.
1. Plasmid preparation
<ol>
	<li>Inoculate 200 mL LB medium containing 100 &#181;g/mL ampicillin with transformed Escherichia coli JM109 incorporating a plasmid encoding for the full-length HEV gt3 Kernow-C1p6 sequence (pBluescript_SK_HEVp6 [JQ...

Starting with the plasmid preparation, DNA yields should exceed 150 ng/&#181;L to be able to perform&#160;multiple linearization from the same plasmid stock, which minimizes the risk of bacteria-induced mutagenesis of crucial genome sequences. Furthermore, it is important to check the restriction digest for the complete plasmid linearization by gel electrophoresis (Figure 8b). A lack of linearized plasmid DNA would induce rolling circle amplification causing the in vitro transcription to be ...

Hepatitis E is a fairly&#160;underestimated disease with increasing prevalence worldwide. About 20 million infections result in more than 70,000 deaths per year1. The underlying agent, the Hepatitis E virus (HEV), was reassigned recently and is now classified within the family Hepeviridae including the genera Orthohepevirus and Piscihepevirus. HEV of various origins are classed within the species Orthohepevirus A-D including isolates from humans, swine, rabbits, rats, birds and other mammals2. At present, eight different genotypes (GT) of the positive-orientated, single-stranded RNA virus have been identified2. Although they differ in their sequence identity, routes of transmission and geographical distribution, their genomic structure is highly conserved. More specific, the 7.2 kbp HEV genome is divided into 3 major open reading frames (ORF1-3). While ORF1 encodes all enzymes needed for a successful replication within the host cell, ORF2 encodes the capsid protein, and the ORF3 protein operates as a functional ion channel required for assembly and release of infectious particles3. Once released into the basal or apical lumen HEV exists in both, quasienveloped and non-enveloped/naked species depending on whether the virus originates from blood or feces, respectively4,5.
While GT1 and GT2 are mainly found in developing countries solely infecting humans6 via the fecal-oral route, GT3, GT4 and GT7 predominantly occur in developed countries1,7 with a variety of species serving as reservoirs, e.g., swine8, rat9, chicken10,11, deer12, mongoose13, bat14, rabbit15,16, wild boar17 and many more7,18,19, providing evidence of zoonosis7,20,21,22. In addition to inadequate sanitary conditions23 and contaminated food products12,24,25,26, transmission via blood transfusion and organ transplantations is also possible27,28. HEV is a common cause of liver cirrhosis and liver failure29 especially in patients with pre-existing liver disease, immunocompromised individuals (genotype 3, 4 and 7) and pregnant women (genotype 1). Of note, there are also extrahepatic manifestations such as hematopoietic disease30,31,32, neurological disorders33 and renal injury34.
To date, the off-label drug ribavirin (RBV) is the treatment of choice for many infected patients35,36. However, cases of treatment failure and poor clinical long-term outcomes have been reported. Treatment failure has been linked to viral mutagenesis and increased viral heterogeneity in chronically infected patients37,38,39. On the contrary, a recent European retrospective multicenter study was not able to correlate polymerase mutations to RBV treatment failure40. In clinical observations and in vitro experiments, interferon41,42,43, sofosbuvir44,45, zinc salts46 and silvestrol47,48 have also shown antiviral effects. Nonetheless, a specific HEV treatment remains to be found, hampered by the lack of knowledge about the HEV life cycle and its pathogenesis. Therefore, a robust cell culture system for virological studies and the development of new antiviral drugs is urgently needed49.
Unfortunately, like other hepatitis viruses, HEV is difficult to propagate in conventional cell lines and usually progresses very slowly leading to low viral loads. Nevertheless, some groups were able to boost viral loads by the generation of cell line subclones50 or the adjustment of media supplements51. Recently the generation of cDNA clones52 and the adaption of primary patient isolates by passaging53,54 further improved HEV propagation in cell culture55. In this protocol, we used the genome of a cell culture adapted Kernow-C1 strain (referred to as p6_WT)54&#160;and a mutant strain harboring a replication-enhancing mutation (referred to as p6_G1634R)37. Kernow-C1 is the most frequently used strain in HEV cell culture and is capable to produce high viral loads. By assessing viral RNA copy numbers, HEV replication can be monitored in vitro. Nevertheless, these techniques do not allow assessment of the number of infectious particles being produced. Therefore, we have established an immunofluorescence staining to determine Focus Forming Units (FFU/mL).
The here described method56 can be used to produce full-length infectious viral particles that are capable to infect a variety of cell types from diverse origins including primary cells and mammalian cell lines. This is a fundamental prerequisite to decipher important aspects of HEV infection and tropism. There is no need for inoculation with usually limited patient isolates. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol comparably efficient. In addition, this system can be used for reverse genetics enabling the study of in vivo identified genome alteration and their impact on HEV replication and fitness. This technique overcomes many limitations and, can path the way for drug development, mutagenesis studies and the evaluation of virus-host interactions such as restriction or entry factors.

<table>
	<tbody>
		<tr>
			<td>0.45 &#181;m mesh</td>
			<td>Sarstedt</td>
			<td>83.1826</td>
			<td>Harvest extracellular Virus</td>
		</tr>
		<tr>
			<td>4 % Histofix</td>
			<td>CarlRoth</td>
			<td>P087.4</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>CarlRoth</td>
			<td>6755.1</td>
			<td>Collagen working solution</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15</td>
			<td>Merck Millipore</td>
			<td>UFC910024</td>
			<td>Virus harvesting</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A1593</td>
			<td>Selection of transformed bacteria</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Roche</td>
			<td>11140965001</td>
			<td>in vitro transcription and electroporation</td>
		</tr>
		<tr>
			<td>BioRender</td>
			<td>BioRender</td>
			<td></td>
			<td>Figure Generation</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Roth</td>
			<td>5239.2</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>Collagen R solution 0.4 % sterile</td>
			<td>Serva</td>
			<td>47256.01</td>
			<td>Collagen working solution</td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Roche</td>
			<td>11140922001</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Biorad</td>
			<td>165-2088</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D21490</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>gibco</td>
			<td>41965-039</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Promega</td>
			<td>M6101</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Promega</td>
			<td>included in P2077</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Roth</td>
			<td>3054.3</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>Escherichia coli JM109</td>
			<td>Promega</td>
			<td>L2005</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>gibco</td>
			<td>10270106</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>GenePulser Xcell Electroporation System</td>
			<td>BioRad</td>
			<td>1652660</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>gibco</td>
			<td>15710049</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Roche</td>
			<td>11140957001</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Braun</td>
			<td>184238001</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Invitrogen</td>
			<td>15630-03</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>gibco</td>
			<td>16050122</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Roth</td>
			<td>P749.1</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Roth</td>
			<td>6781.3</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth</td>
			<td>3904.2</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>gibco</td>
			<td>25030081</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>L-Glutathione reduced</td>
			<td>Sigma-Aldrich</td>
			<td>G4251-5G</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>gibco</td>
			<td>31095-029</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>MEM NEAA (100&#215;)</td>
			<td>gibco</td>
			<td>11140-035</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Roth</td>
			<td>2189.2</td>
			<td>Cytomix</td>
		</tr>
		<tr>
			<td>Microvolume UV-Vis spectrophotometer NanoDrop One</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td>DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>MluI enzyme</td>
			<td>NEB</td>
			<td>R0198L</td>
			<td>Linearization</td>
		</tr>
		<tr>
			<td>NEB buffer</td>
			<td>NEB</td>
			<td>included in R0198L</td>
			<td>Linearization</td>
		</tr>
		<tr>
			<td>NucleoSpin Plasmid kit</td>
			<td>Macherey &#38; Nagel</td>
			<td>740588.250</td>
			<td>Plasmid preparation</td>
		</tr>
		<tr>
			<td>NucleoSpin RNA Clean-up Kit</td>
			<td>Macherey &#38; Nagel</td>
			<td>740948.250</td>
			<td>RNA purification</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>gibco</td>
			<td>70011051</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Plasmid encoding full-length HEV genome (p6_G1634R)</td>
			<td>Todt et.al</td>
			<td></td>
			<td>Virus production</td>
		</tr>
		<tr>
			<td>Plasmid encoding full-length HEV genome (p6_WT)</td>
			<td>Shukla et al.</td>
			<td>GenBank accession no. JQ679013</td>
			<td>Virus production</td>
		</tr>
		<tr>
			<td>Primary antibody 1E6</td>
			<td>LS-Bio</td>
			<td>C67675</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Primary antibody 8282</td>
			<td></td>
			<td></td>
			<td>Rainer Ulrich, Friedrich Loeffler Institute, Germany</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>DNA extraction</td>
		</tr>
		<tr>
			<td>Ribo m7G Cap Analog</td>
			<td>Promega</td>
			<td>P1711</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>RNase away</td>
			<td>CarlRoth</td>
			<td>A998.3</td>
			<td>RNA purification</td>
		</tr>
		<tr>
			<td>RNasin (RNase inhibitor)</td>
			<td>Promega</td>
			<td>N2515</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>Secondary antibody donkey anti-mouse 488</td>
			<td>Thermo Fisher</td>
			<td>A-21202</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Secondary antibody goat anti-rabbit 488</td>
			<td>Thermo Fisher</td>
			<td>A-11008</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Sodium Pyruvat</td>
			<td>gibco</td>
			<td>11360070</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>Promega</td>
			<td>P2077</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>Transcription Buffer</td>
			<td>Promega</td>
			<td>included in P2077</td>
			<td>in vitro transcription</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>CarlRoth</td>
			<td>3051.3</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5 %)</td>
			<td>gibco</td>
			<td>15400054</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>ultra-low IgG</td>
			<td>gibco</td>
			<td>1921005PJ</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Roche</td>
			<td>11140949001</td>
			<td>in vitro transcription</td>
		</tr>
	</tbody>
</table>

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.

In this protocol, we describe the production of high titer infectious HEVcc. The first step is to isolate plasmid DNA (pBluescript_SK_HEVp654 and pBluescript_SK_HEVp6-G1634R37, Figure 8a), which then is linearized by restriction digestion and purified for in vitro transcription (Figure 1). A successful linearization can be verified by comparing the non-digested plasmid-DNA to the digested plasmid-DNA using gel elec...

Watch this Scientific Journal Video about A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks at JoVE.com

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

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 ...

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