The overall goal of this procedure is to determine the capacity of sera collected from Zika virus infected rhesus macaques to mediate antibody dependent enhancement of dengue virus infection in vitro. This method can help answer key questions related to antibody dependent enhancement of infection of dengue caused by Zika virus induced cross-reactive antibodies. The main advantage of this technique is that it is high troper, simple, and easy to perform.
Demonstrating this procedure will be William Valiant, an emerging infectious disease graduate student, in my laboratory. Begin by thawing the serum samples at room temperature. When thawed, transfer 100 microliters of each serum sample to a sterile tube and heat and activate for 30 minutes at 56 degrees Celsius.
Next, make 10-fold serial dilutions of the serum sample ranging from one-to-one, to one-to-one thousand in cold RPMI 10. Then transfer 10 microliters of each diluted serum sample to the wells of a sterile 96 well V-bottom plate. Next, thaw dengue one, two, three, and four proporter viral particles in a 37 degrees Celsius water bath, ensuring a volume of approximately 170 microliters of each RVP per serum sample.
Once thawed, immediately transfer the tubes of RVPs to ice. Then pipet 10 microliters of RVPs into the wells containing serum sample dilutions into the no serum control wells. Mix thoroughly by pipetting up and down five to ten times.
Transfer the 96 well V-bottom plate to an incubator and incubate for an hour at 37 degrees Celsius, in the presence of 5%carbon dioxide. While the 96 well V-bottom plate is incubating, clean the surface of the bio-safety cabinet with 70%ethanol, and run the UV light for 15 minutes. Then remove a fully confluent T-75 flask of K562 cells from the incubator.
Mix the cells well using a sterile five millimeter pipet and transfer five milliliters to a sterile 15 milliliter conical tube. Next, remove 10 microliters of cells from the tube, mix with 10 microliters of trypan blue and load into a hemocytometer. Count the cells and calculate the total number of cells.
After centrifuging the cells at 12 hundred times G for 10 minutes, decant the supernatant and re-suspend the cells in warm RPMI 10 at a concentration of 80 thousand cells per 30 microliters of media. After removing the 96 well V-bottom plate from the incubator, transfer 30 microliters of K562 cells to each wall of the 96 well V-bottom plate. And mix thoroughly by pipetting up and down five to ten times.
Then return the plate to the incubator for one hour. Following the incubation, centrifuge the plate at 12 hundred times G for five minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10%bleach.
Then wash the cells in each wall by re-suspending them in 125 microliters of warm RPMI 10 and centrifuging as before. After decanting the last wash, add 100 microliters of warm RPMI 10 to each well. Mix up and down with the pipet and incubate the plate for 48 hours at 37 degrees Celsius in the presence of 5%carbon dioxide.
Two days later, remove the plate from the incubator and transfer to a bio-safety cabinet. Using a multichannel pipet, mix the contents of each well and transfer to 96 well U-bottom plate. Rinse each well of the 96 well V-bottom plate with 100 microliters of 1%paraformaldehyde and PBS and transfer the remaining cells and PBS to the respective wells in the 96 well U-bottom plate.
Mix thoroughly using a multichannel pipet. Cover the plate with aluminum foil and let it sit in the incubator for 30 minutes to fix the cells. Prepare the flow cytometer by running unstained K562 cells to calibrate the side and forward scatter settings using the ethyl-one channel for GFP florescence.
Then acquire 30 thousand to 50 thousand cells from each sample. Using flow cytometry analysis software, set the first gate based on the forward scatter area versus forward scatter height to include single cells and exclude autofluorescent dublets from analysis. Calculate the average percentage of GFP positive cells for each condition and then calculate fold enhancement of infection for each serum dilution.
Graph fold enhancement on the y-axis versus dilution on the x-axis and perform statistical analysis using ANOVA followed by Tukey's post-hoc test for multiple comparisons. Data from a representative animal using dengue virus one reporter virus particles are shown here. The percentage of GFP positive cells for the no serum control sample was 0.253%The undiluted serum sample was 2.58%GFP positive.
The proportion of GFP positive cells reached a high of 12.8%at the one to ten serum dilution following to 0.735%at a one to one hundred dilution and 0.022%at a one to one thousand dilution. Antibody dependent enhancement of dengue infection was examined for the four dengue virus sera types. The data shows that the serum collected 16 weeks post-infection significantly enhanced infection of K562 cells by all dengue virus sera types at a dilution of one to ten.
Once mastered, this technique can be done in 48 hours if it is performed properly. While attempting this procedure, it is important to remember to acquire the data within one to two hours after the final fixation step of 30 minutes is completed. Don't forget that working with infected serum can be extremely hazardous and precautions such as PPE, and BSL2 Level practices should always be taken while preforming this procedure.
