This study investigates the survival kinetics of viruses by measuring host-cell impedance. The methodology involves assessing the effects of viral infection on host cells and their morphological changes over time.
Viruses bind to the host-cell receptor and internalize into an endosome. The endosomal membrane fuses with the viral membrane, releasing viral contents inside the cell.
Upon reaching the nucleus, the viral genome transcribes into mRNA and later translates to form viral proteins. The viral components enter the cytoplasm and assemble into virions, which pinch out of the cell and cause cytopathic effects ― changes in the host cell's morphology, and death.
To assess virus survival kinetics by measuring host-cell impedance, take a multiwell plate coated with impedance-measuring microelectrodes at its bottom. Add a host-cell suspension to the wells, and incubate to allow the cells to adhere to the electrode surface, and grow.
Next, take saline pre-treated viral suspensions with different viral loads. Saline treatment supplies the virus with chloride, which produces hypochlorous acid that kills it ― reducing the viral infectivity. Add these viral suspensions into their respective wells.
Measure the cellular impedance ― a phenomenon of opposing the flow of electric current through the electrodes ― by the adherent cells.
Viruses infect adherent host cells and change their morphology, causing the detachment of the morphed cells from the electrodes. Over time, as more cells get infected, the host cell-mediated cellular impedance decreases.
Plot the cell index, which is the change in impedance due to infected cell detachment, over time.
To assess the influenza A virus survival kinetics, add sodium chloride to a final concentration of 35 grams per liter in distilled water, and add 900 microliters of the resulting saline solution to 2-milliliter cryotubes. Add 100 microliters viral stock to each cryotube, and place the tubes in the 35-degree Celsius incubator, for the appropriate experimental time period.
The day before the end of the incubation, seed 3 x 104 freshly-split MDCK cells in 100 microliters of virus propagation medium per well, in a 16-well microtiter plate, and repeat steps allowing measurement of the background impedance and uniform distribution of the cells onto the bottom of the wells. Then, grow the cells for 24 hours at 37 degrees Celsius and 5% carbon dioxide.
The next day, wash the cells twice, then, infect the cells with 100 microliters of the saline-distilled virus, diluted 10 times in fresh culture medium, as demonstrated, and monitor the cell impedance every 15 minutes for at least 100 hours.
繁體中文
