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 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.
Hepatitis E is a fairly 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 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.
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
2. Linearization and DNA purification
Figure 1: Schematic experimental setup for the plasmid linearization and DNA purification. Please click here to view a larger version of this figure.
NOTE: The linearization increases RNA yield during in vitro transcription (step 3)
3. In-vitro transcription of full-length HEV genotype 3 p6 DNA and RNA purification
Figure 2: Schematic experimental setup for the in vitro transcription and RNA purification. Please click here to view a larger version of this figure.
NOTE: In vitro transcription is necessary to produce viral genomic RNA from plasmid DNA.
4. Preparation of HepG2 cells for cell culture derived HEV (HEVcc) production
Figure 3: Schematic experimental setup for the preparation of HepG2 cells for cell culture derived HEV (HEVcc) production. Please click here to view a larger version of this figure.
NOTE: To avoid contamination, preparation of cells, electroporation, infection, harvesting and cell fixation were carried out under sterile conditions in a biosafety level 2 facility. Incubation steps at 37 °C that involve cells were accomplished in a 5% CO2 incubator.
5. Electroporation of HepG2 cells
Figure 4: Schematic experimental setup for the electroporation of HepG2 cells. Please click here to view a larger version of this figure.
6. Harvesting of intra- and extracellular HEVcc
Figure 5: Schematic experimental setup for harvesting intra- and extracellular HEVcc. Please click here to view a larger version of this figure.
7. Infection of HepG2/C3A cells with intra- and extracellular HEVcc
Figure 6: Schematic experimental setup for the infection of HepG2/C3A cells with intra- and extracellular HEVcc. Please click here to view a larger version of this figure.
NOTE: The infection of HepG2/C3A cells shall ensure that infectious particles were produced. Additionally, the titration of the harvested intracellular and extracellular HEVcc is used to calculate the virus titers in FFU/mL. This will be later referred to as infection control (IC).
8. Immunofluorescence staining of transfection- and infection control
Figure 7: Schematic experimental setup for the immunofluorescence staining of transfection and infection control. Please click here to view a larger version of this figure.
9. FFU determination
NOTE: One FFU is defined as one or more ORF2-positive cells separated from another FFU by at least three negative cells.
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 electrophoresis. In addition to a size-shift, only one DNA band should be visible representing the linear form. The linearization is complete when the two other bands above and below the linear form, representing the nicked circle and the supercoiled form, respectively, are completely diminished (Figure 8b). The yield of the purified DNA should exceed 150 ng/µL. Only if these characteristics hold true the linearized DNA should be used for in vitro transcription. The in vitro transcribed RNA should be as well checked using gel electrophoresis and in case of low RNase abundancy should show distinct bands rather than a blurred smear (Figure 8c). Additionally, the purified RNA yield should exceed 500 ng/µL The in vitro transcribed RNA (Figure 2) eventually is electroporated into HepG2 cells for virus production (Figure 3 and Figure 4). Successful electroporation is monitored by the immunofluorescence staining of the transfection control (Figure 9a). The transfection efficiency should exceed 40% (Figure 9b). A replication deficient mutant serves as a negative control, to ensure specificity of the ORF2 staining, as no ORF2 expression is expected (Figure 9c). After 7 days of incubation the enveloped (extracellular) HEVcc are harvested by collecting and filtering the cell culture supernatant. Non-enveloped (intracellular) HEVcc is released from the cells by several freeze and thaw cycles. To remove any cell debris the cell lysate is centrifuged at high speed (Figure 5). Subsequently, both HEVcc species are utilized to infect HepG2/C3A cells by serial dilution (Figure 6 and Figure 9d). According to the equations above (see step 9) viral titers are determined by FFU calculation.
Representative results are depicted in Figure 9. The transfection control should comprise around 50% ORF2-positive cells to guarantee an efficient amount of virus being produced (Figure 9a,b). The less ORF2-positive cells the lower the titer will be. Precisely following the steps mentioned in the protocol will generate titers that vary between 105 and 3 x 106 FFU/mL for the non-enveloped (intracellular) HEVcc. For the enveloped (extracellular) HEVcc titers between 102 and 5 x104 FFU/mL are expected (Figure 9e). Additionally, elevated FFU counts were observed for the G1634R mutant. When calculating the ratio between genome copies and infectious viral particles the produced intracellular HEVcc for both p6_WT and p6_G1634R was found to be lower compared to the extracellular HEVcc, suggesting a higher specific infectivity of the non-enveloped HEV species (Figure 9f).
Figure 8: Generation of in vitro transcribed RNA.
(A) Example of an absorbance spectrum of isolated Plasmid-DNA. (B) Gel electrophoresis of non-digested and digested Plasmid-DNA. (C) Gel electrophoresis of in vitro transcribed RNA. Please click here to view a larger version of this figure.
Figure 9: Representative results of high titer HEVcc production.
(A) Transfection control of electroporated HepG2 cells. Transfected cells were stained with anti-ORF2 antibody (green, LS-Bio) and DAPI (blue). (B) The transfection efficiency was calculated by the number of ORF2-positive cells normalized to the number of total cells. (C) A replication-deficient mutant serves as a negative control for immunofluorescence staining, to ensure ORF2 specificity. (D) For FFU determination serial dilutions of the produced virus stocks were performed. ORF2 positive cells are depicted in white. (E) Viral titers were calculated by FFU counting and (F) viral loads were determined by qPCR and normalized to FFU/mL. Bars show the mean and standard deviation of 38 and 10 independent experiments, respectively. Please click here to view a larger version of this figure.
Working solution | Concentration | Volume |
Collagen | 40 mL | PBS |
40 µl | Acetic acid | |
1 mL | Collagen R solution 0.4% sterile | |
Cytomix | 120 mM | KCl |
0.15 mM | CaCl2 | |
10 mM | K2HPO4 (pH 7.4) | |
25 mM | HEPES | |
2 mM | EGTA | |
DMEM complete | 500 mL | DMEM |
5 mL | Pen/Strep | |
5 mL | MEM NEAA (100X) | |
5 mL | L-Glutamin | |
50 mL | Fetal bovine serum | |
MEM complete | 500 mL | MEM |
5 mL | Sodium Pyruvat | |
5 mL | Gentamycin | |
5 mL | MEM NEAA (100X) | |
5 mL | L-Glutamine | |
50 mL | ultra-low IgG |
Table 1: Table of Buffer Composition.
Starting with the plasmid preparation, DNA yields should exceed 150 ng/µL to be able to perform 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 less efficient. In addition, RNA integrity should be confirmed to evaluate the abundance of RNases within the sample (Figure 8c). Only then an electroporation of target cells should be considered. Of note, before starting with the preparation of HepG2 cells, make sure everything is at hand and well-prepared avoiding long waiting periods. Especially, assure short-time storage of the cells and RNA in the Cytomix until electroporation. To circumvent heating of the cells during electroporation it is essential to cool the buffer on ice beforehand. The duration of the pulse should not exceed 20-25 ms and 270 V. After electroporation the cells require a quick transfer into fresh medium to ensure cell viability. Before infection of target cells, the TC should be checked for the percentage of ORF2-positive cells. In case the TC shows no ORF2-positive cells it is most likely that the electroporation has failed or the staining did not work, and the experiment should be repeated.
When harvesting intracellular HEVcc special attention should be payed to the speed of the freeze and thaw cycles. Viral titers can be increased by executing the freezing in liquid nitrogen rather than at -80 °C. On the contrary, the thawing should take place the slowest way possible suggesting the storage on ice until the cell suspension is liquidated completely. Nonetheless, it is also possible to thaw the cell suspension at room temperature, in a 37 °C incubator or water bath, however the faster the thawing will be executed the more the titers will decrease.
Depending on the following experiments it is also possible to do the freeze and thaw cycles in MEM complete or 1x PBS without dramatic loss of viral titers, however, one should bear in mind that this causes the extracellular HEVcc to be in a different medium than the intracellular HEVcc.
Following these crucial steps of the protocol, expected titers vary between 105 and 3 x 106 FFU/mL for the non-enveloped (intracellular) HEVcc. For the enveloped (extracellular) HEVcc titers between 102 and 5 x 104 FFU/mL are awaited (Figure 9e). So far, studies employing HEV cell culture systems monitor viral replication and propagation predominantly by qPCR. The assessment of RNA genome copies yet provides no insight into assembly and release of infectious particles.
A previous study58 successfully propagated patient isolates in cell culture with maximum titers of 103 TCID50/mL. As shown recently, it is also possible to successfully passage HEVcc in cell culture and adapt our protocol to other strains, such as 47832c and 83-2 which do not harbor an insertion in the hypervariable region56. Also, whether patient derived sequences can be cloned into the Bluescript vector backbone and still yield high viral titers was not tested. With the introduced method, non-enveloped as well as enveloped viral particles can be harvested and used to inoculate a variety of naïve cell lines such as A549, Huh7.5, Jeg-3 and primary human and porcine hepatocytes, providing a benefit for future applications such as the investigation of HEV tropism, pathogenesis, drug development, viral and host interactions, inactivation studies, neutralizing antibodies and many more.
The authors have nothing to disclose.
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.
0.45 µm mesh | Sarstedt | 83.1826 | Harvest extracellular Virus |
4 % Histofix | CarlRoth | P087.4 | Immunofluorescence |
Acetic acid | CarlRoth | 6755.1 | Collagen working solution |
Amicon Ultra-15 | Merck Millipore | UFC910024 | Virus harvesting |
Ampicillin | Sigma-Aldrich | A1593 | Selection of transformed bacteria |
ATP | Roche | 11140965001 | in vitro transcription and electroporation |
BioRender | BioRender | Figure Generation | |
CaCl2 | Roth | 5239.2 | Cytomix |
Collagen R solution 0.4 % sterile | Serva | 47256.01 | Collagen working solution |
CTP | Roche | 11140922001 | in vitro transcription |
Cuvette | Biorad | 165-2088 | Electroporation |
DAPI | Invitrogen | D21490 | Immunofluorescence |
DMEM | gibco | 41965-039 | Cell culture |
DNAse | Promega | M6101 | in vitro transcription |
DTT | Promega | included in P2077 | in vitro transcription |
EGTA | Roth | 3054.3 | Cytomix |
Escherichia coli JM109 | Promega | L2005 | Transformation |
Fetal bovine serum | gibco | 10270106 | Cell culture |
Fluoromount | SouthernBiotech | 0100-01 | Immunofluorescence |
GenePulser Xcell Electroporation System | BioRad | 1652660 | Electroporation |
Gentamycin | gibco | 15710049 | Cell culture |
GTP | Roche | 11140957001 | in vitro transcription |
H2O | Braun | 184238001 | Immunofluorescence |
Hepes | Invitrogen | 15630-03 | Cytomix |
Horse serum | gibco | 16050122 | Immunofluorescence |
K2HPO4 | Roth | P749.1 | Cytomix |
KCL | Roth | 6781.3 | Cytomix |
KH2PO4 | Roth | 3904.2 | Cytomix |
L-Glutamin | gibco | 25030081 | Cell culture |
L-Glutathione reduced | Sigma-Aldrich | G4251-5G | Cytomix |
MEM | gibco | 31095-029 | Cell culture |
MEM NEAA (100×) | gibco | 11140-035 | Cell culture |
MgCl2 | Roth | 2189.2 | Cytomix |
Microvolume UV-Vis spectrophotometer NanoDrop One | Thermo Fisher | ND-ONE-W | DNA/RNA concentration |
MluI enzyme | NEB | R0198L | Linearization |
NEB buffer | NEB | included in R0198L | Linearization |
NucleoSpin Plasmid kit | Macherey & Nagel | 740588.250 | Plasmid preparation |
NucleoSpin RNA Clean-up Kit | Macherey & Nagel | 740948.250 | RNA purification |
PBS | gibco | 70011051 | Cell culture |
Pen/Strep | Thermo Fisher | 15140122 | Cell culture |
Plasmid encoding full-length HEV genome (p6_G1634R) | Todt et.al | Virus production | |
Plasmid encoding full-length HEV genome (p6_WT) | Shukla et al. | GenBank accession no. JQ679013 | Virus production |
Primary antibody 1E6 | LS-Bio | C67675 | Immunofluorescence |
Primary antibody 8282 | Rainer Ulrich, Friedrich Loeffler Institute, Germany | ||
QIAprep Spin Miniprep Kit | Qiagen | 27106 | DNA extraction |
Ribo m7G Cap Analog | Promega | P1711 | in vitro transcription |
RNase away | CarlRoth | A998.3 | RNA purification |
RNasin (RNase inhibitor) | Promega | N2515 | in vitro transcription |
Secondary antibody donkey anti-mouse 488 | Thermo Fisher | A-21202 | Immunofluorescence |
Secondary antibody goat anti-rabbit 488 | Thermo Fisher | A-11008 | Immunofluorescence |
Sodium Pyruvat | gibco | 11360070 | Cell culture |
T7 RNA polymerase | Promega | P2077 | in vitro transcription |
Transcription Buffer | Promega | included in P2077 | in vitro transcription |
Triton X-100 | CarlRoth | 3051.3 | Immunofluorescence |
Trypsin-EDTA (0.5 %) | gibco | 15400054 | Cell culture |
ultra-low IgG | gibco | 1921005PJ | Cell culture |
UTP | Roche | 11140949001 | in vitro transcription |