JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:52 min • August 31st, 2026
Begin with a culture of human liver cancer cells transfected with hepatitis E virus (HEV) RNA to produce viral particles.
Some HEVs acquire a host-derived endosomal membrane and exit the cell, while others remain within the cell as non-enveloped HEVs.
To harvest extracellular HEVs, collect and filter the culture supernatant to remove cellular debris.
Store at an ultra-low temperature to preserve infectivity.
To harvest intracellular HEVs, wash the cells with a buffer.
Add trypsin-EDTA to detach the cells, then add a medium to stop enzymatic activity.
Transfer the suspension into a tube and centrifuge.
Discard the supernatant containing residual trypsin. Resuspend the pellet and transfer it to a tube.
Freeze the cells in liquid nitrogen and thaw them on ice.
Repeat the freeze-thaw cycles to lyse the cells, releasing the viruses.
Centrifuge, collect the supernatant containing the viruses, and store at an ultra-low temperature.
On day six, filter the supernatant through a 0.45 micrometer 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.