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

Hepatitis E virus research has been hampered by the lack of an efficient cell culture system. Our techniques overcome these limitations paving the way to drug design and vaccine development. With this protocol, we are able to produce infectious high titer viruses of both non-envelope and envelope HEV particles, facilitating the infection of a variety of cells.

Execution of this protocol requires access to a BSA-2 facility and experience with basic cell culture techniques. To linearize the plasmid DNA, mix 10 micrograms of the template DNA with 10 microliters of buffer and two microliters of the Micrococcus luteus restriction enzyme and adjust the final volume to 100 microliters with water. Then incubate the solution for one hour at 37 degrees Celsius and confirm linearization of the plasmid by agarose gel electrophoresis according to standard protocols.

After plasmid DNA isolation and linearization, the success of the linearization can be verified by comparing the non-digested plasmid DNA to the digested plasmid DNA by gel electrophoresis. For in vitro transcription of the purified, full-length hepatitis E virus DNA, mix two micrograms of the linearized DNA template with the appropriate reagents and use nucleus-free water to bring the final volume of the solution to 100 microliters. After thorough mixing, incubate the solution for two hours at 37 degrees Celsius.

After two hours, add an additional two microliters of T7 RNA polymerase to the tube. Then mix the reaction and incubate the tube at 37 degrees Celsius for another two hours. At the end of the incubation, digest the initial DNA template with 7.5 microliters of DNAs with thorough mixing and incubate for 37 degrees Celsius for 30 minutes.

After the extraction, carefully check the RNA integrity and yield by agarose gel electrophoresis according to standard protocols. In the case of low RNAs abundancy, distinct bands, rather than a blurred smear should be observed. Some steps require quick execution and therefore thorough preparation.

Also, pay special attention to RNA integrity and cell viability. To prepare human liver cancer cells for cell culture-derived hepatitis E virus production, seed the cells in 20 milliliters of complete DMEM onto a 15 centimeter collagen coated culture dish and incubate at 37 degrees Celsius until they reach 90%confluency. At the end of the culture, wash the cells with 10 milliliters of PBS before detaching them from the cell culture plate with three milliliters of 0.05%Trypsin-EDTA at 37 degrees Celsius.

Resuspend the detached cells in 10 milliliters of complete DMEM and transfer the cells into a 50 milliliter conical tube for counting. Transfer five times 10 to the six cells to a new 50 milliliter tube and wash the cells two times with 35 milliliters of PBS per wash. After the second wash, place the cells on ice without removing the supernatant.

For electroporation of the target cells, supplement 384 microliters of cytomix with two millimolar ATP and five millimolar glutathione, subsequently store on ice until further usage. Carefully replace the supernatant with the entire 400 microliters of solution. Add five micrograms of purified viral genomic RNA to the cells and transfer the cell suspension to a four millimeter cuvette for electroporation at 975 microfarad and 270 volts for 20 milliseconds.

After electroporation, use a Pasteur pipette to transfer the cells as quickly as possible into 11 milliliters of complete DMEM and seed 10 milliliters of the electroporated cells into a 10 centimeter culture dish coated with collagen. Next, add 1.3 times 10 to the fifth cells in 300 microliters of medium evenly onto a cover slip in one well of a collagen-coated 24 microtiter plate and place the plate and dish in the cell culture incubator for 24 hours. The next day, replace the supernatant in the dish with 10 milliliters of fresh DMEM and return the dish to the cell culture incubator for an additional six days.

On day five to seven, depending on the cell density, perform immunofluorescent staining of the transfection control to allow calculation of the number of cells expressing the target protein of interest normalized to the total number of cells. Successful electroporation can be monitored by immunofluorescent staining of the transfection control. The transfection efficiency should exceed 40%A replication deficient mutant can serve as a negative control to confirm the specificity of the staining.

To harvest extracellular cell culture-derived hepatitis E virus particles, on day six, filter the supernatant through a 0.45 microliter mesh to remove any cell debris and store the harvested extracellular cell culture-derived hepatitis E virus at four degrees Celsius. For intracellular cell culture-derived hepatitis E virus particle harvest, wash cells with PBS and detach the cells with 1.5 milliliters of 0.05%Trypsin-EDTA at 37 degrees Celsius. When the cells have detached, stop the reaction with 8.5 milliliters of DMEM and transfer the cell suspension to a 50 milliliter tube for centrifugation.

Add 1.6 milliliters of DMEM complete medium per electroporation to individual two milliliter reaction tubes and use 800 microliters of the medium to resuspend the cells, thus transferring the cells to the tubes. Freeze the cells out in liquid nitrogen, and subsequently thaw the cells on ice three times before high speed centrifuging the resulting lysates for 10 minutes at 10, 000 times g. Then transfer the supernatants into new tubes without disturbing the cell debris pellets for minus 80 degrees Celsius storage.

To assess the titers of the newly produced intra and extracellular hepatitis E virus stocks, seed two times 10 to the four cells per 100 microliters of MEM per well into the 60 center wells of a collagen-coated 96-well microtiter plate and fill the outermost wells with 100 microliters of PBS per well. After 24 hours at 37 degrees Celsius, add 50 microliters of extracellular virus particle supernatant to the first row of cells. After thoroughly mixing, serially dilute the well contents six times at a one to three concentration per well by transferring 50 microliters of supernatant from the previous to the next row of cells in duplicate until the last row of cells.

To infect the cells with intracellular cell culture-derived hepatitis E virus particles, add 25 microliters of the intracellular virus particle supernatant to the top row of cells and mix well before serially diluting the cells six times at a one to five ratio with 25 microliters of dilution in duplicate as demonstrated. Then place the plate in the cell culture incubator for seven days before immunofluorescent staining according to the protocol in the manuscript. Following incubation and immune staining, the plates can be analyzed using immunofluorescence microscopy.

After immunostaining the final titer of the intra and extracellular particles can be calculated using the appropriate formula as indicated. After the target cell infection with cell culture-derived E virus particles by serial dilution, titers between one times 10 to the fifth and three times 10 to the six focus forming units per milliliter can be expected from the cell cultures infected with the non-enveloped intracellular cell culture-derived hepatitis E virus particles. In cultures infected with enveloped extracellular cell culture-derived hepatitis E virus particles, titers between one times 10 to the second and one times 10 to the fourth focus forming units per milliliter are expected.

In addition, the ratio between genome copies and non-enveloped intracellular infectious virus particles is typically lower than that observed for extracellular cell culture-derived hepatitis E virus particle infected cultures, suggesting a higher specific infectivity of the non-enveloped hepatitis E virus species. Viral particle production and target cell infection require a full viral life cycle, and can provide new insights into virus-host interactions. This protocol can be modified to perform replication kinetics or to produce particles that can be used in infection assays.

Most of our research currently depends on infectious particles, both in the enveloped and non-enveloped HEV. And articles using our techniques are currently being published.