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

Summary

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.

Abstract

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.

Introduction

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 identified

Protocol

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

1. Inoculate 200 mL LB medium containing 100 µg/mL ampicillin with transformed Escherichia coli JM109 incorporating a plasmid encoding for the full-length HEV gt3 Kernow-C1p6 sequence (pBluescript_SK_HEVp6 [JQ679013]⁵⁴ or pBluescript_SK_HEVp6-G1634R³⁷). Incubate for 16 h at 37 °C under permanent agitation (170 rpm)....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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