The overall goal of this study is to establish a mammalian cell-based in vitro Zika virus infectious culture system to study Zika virus replication, as well as utilization of the in vitro platform for drug development. This step-by-step detailed protocol of Zika virus infectious cell culture system can help address the mechanism of Zika virus mediated disease pathogenesis and evolution of viral virulence. The main advantage of this technique is that it can allow for large-scale production of Zika virus for drug and vaccine development.
Begin by harvesting Vero cells at 80%cell density in a T75 culture flask by aspirating the medium from the flask and rinsing the cells with two milliliters of phosphate-buffered saline. Add two milliliters of 0.25%trypsin and incubate the flask at 37 degrees Celsius for five minutes. Following the incubation, add eight milliliters of growth medium containing serum to inactivate the trypsin.
Then, transfer the cells to a 15 milliliter tube. Next, remove 10 microliters of cells and mix with an equal amount of 0.4%trypan blue. Load the prepared mix to the cell counting chamber slide and obtain account of the viable cells using an automated cell counter.
Seed a total of seven-million cells into a T160 flask in a 30 milliliter volume. The next day, prepare an appropriate multiplicity of infection of Zika virus inoculum in 10 milliliters of serum-free culture medium per flask. Safe handling of the virus is critical to prevent the spread of infection.
Remove the spent medium from the T160 flask and then add the full 10 milliliters of freshly prepared viral inoculum. Incubate the inoculated flasks at 37 degrees Celsius with 5%CO2 for four to six hours. Spread the inoculum by gently tilting the flask sideways every hour of the incubation.
At the completion of the incubation, replace the inoculum with 30 milliliters of warm serum-supplemented growth medium. Then, continue the viral culture for the next 96 hours. After four days, verify the progression of infection by observing the appearance of viral plaques on the cell monolayer, using a phase contrast microscope.
Take images as needed at 40x and 200x magnification. Harvest cell-culture supernatants at the 96 hour time point and centrifuge the supernatant at 300 times G for 10 minutes at four degrees Celsius to remove cell debris. After centrifugation, carefully remove the supernatants without disturbing the pelleted debris, and transfer them into 15 milliliters tubes.
Store the viral culture supernatants in multiple aliquots at negative 80 degrees Celsius. To titer the virus, first seed naive Vero cells into a 12-well plate at one times 10 to the fifth cells per well, in a two milliliter volume. The following day, prepare tenfold serial dilutions of viral culture supernatants, collected from the T160 flask using serum-free medium.
When preparing serial dilutions, a viral culture supernatant for titer acids, make sure to safely handle the virus to prevent infection. Next, remove the spent medium from each well of the 12-well plate containing Vero cells, and add 400 microliters of the diluted inoculum onto Vero cells in triplicate. Incubate the inoculated plates in the tissue-culture incubator for four to six hours.
At the end of the incubation, replace the inoculum with two milliliters of serum-supplemented medium per well. At 48 hours post inoculation, count the viral foci at the appropriate dilutions, using a phase contrast microscope. Calculate the viral titer as plaque-forming units per milliliter.
After inoculating Vero cells in a 12-well plate with high and low titers of Zika virus, and incubating for 48 and 96 hours, genome replication is assessed by quantitative PCR for expression of the Zika virus genome, and by immunocytochemistry for double-stranded RNA. For immunocytochemistry, remove the medium from the wells of the 12-well plate, and add one milliliter of methanol to each well. Allow the cells to fix at four degrees Celsius for 30 minutes.
Next, wash the fixed cells three times with 1x PBS. Then add blocking buffer and incubate for one hour at room temperature. Dilute the mouse monoclonal anti-double-stranded RNA antibody J2 one to 100 in blocking buffer.
Add it to the cells, and incubate the cells in this solution overnight at four degrees Celsius. The next day, dilute goat anti-mouse IgG-594 secondary antibody one to 1, 000 in blocking buffer, and after washing the cells with 1x PBS, add it to the cells, and incubate them for one hour at room temperature. After washing the cells with 1x PBS, stain for nuclei using hoechst dye, and observe the cells using a fluorescent microscope at 100x magnification.
Six hours after seeding a 12-well plate with naive Vero cells, as before, add cytokines of interest at indicated concentrations in biological duplicates. 12 hours posttreatment, perform Zika virus infection, and include negative-control wells without infection. Four hours after infection, replace the viral inoculum with cytokine-treated medium and then incubate the cells at 37 degrees Celsius for an additional 44 hours.
At 48 hours post infection, count viral plaques using a phase contrast microscope, and acquire representative images of infected cells at 40x magnification. This graph shows Zika virus genomic content measured by RT-qPCR, both Pan genotype and PRVABC-59 strain primers showed equal sensitivity and specificity. As expected, hepatitis-C viral primers did not amplify any product.
Zika viral growth kinetics at the indicated time points in low and high titer infected cells, compared to that of the uninfected mock control, are shown here. Results from the immunocytochemistry assay show that at 48-hours post infection, the infected cells are in a few clusters, whereas at 96-hours post infection, most of the cells are infected. This graph shows modulation of Zika viral growth by various cytokines, compared to that of the vehicle control.
These phase-contrast images show Zika virus-infected cells with or without cytokine treatment. Viral plaques formed by infected cells can be seen as foci. Note that interferon treated wells do not have viral plaques.
Following this procedure, other methods such as Zika virus high-resolution domain mapping for viral protein localization and replication in mosquito and human cells can be performed to answer additional questions, such as elucidation of the mechanism of disease pathogenesis and evolution of viral virulence. After watching this video, you should have a good understanding of how to establish the mammalian cell-based in vitro Zika virus infectious culture system to study Zika virus replication, as well as the placation of in vitro platform for drug and vaccine development.
