We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.
This method can be used to evaluate the effects of pre-existing immunity against dengue virus on Zika virus infection, using actual human patient serum and primary human immune cells. The use of actual human patient serum samples and human primary cells provide deeper insight into the antibody dependent enhancement of Zika virus by pre-immune sera and antibodies. This method provides insight into the ability of antibodies within a patient's blood to enhance a virus infection in other human cells.
It is important to ensure that the viral infection levels are high enough for detection but low enough not to overwhelm the cells or to interfere with the data interpretation. Begin by seeding three times ten to the fourth cells in 100 microliters of cell culture medium per well in a sterile, flat bottom 96-well plate. When all the cells have been plated, place the plate in a 37 degree Celsius and 5%carbon dioxide incubator and make 10 fold serial dilutions of thawed human serum samples in serum free medium.
Be sure to keep the serum dilutions and dilution factor constant for all the pre-immune serums before adding the dilutions to the virus solutions and the cells. Next, thaw Zika virus strains stock solution in a 37 degree Celsius water bath for one to two minutes before quickly transferring the stock to ice. Add a 0.1 multiplicity of infection equivalent volume of Zika virus to the serum aliquots and place the plate in the cell culture incubator for one hour to allow the dengue virus antibodies to form complexes with the Zika virions.
At the end of the incubation, wash the cells with 100 microliters of PBS per well and add 50 microliters of immune complex to each well. After the two hour incubation, remove the medium and wash the wells twice with 100 microliters of PBS per well to completely remove the immune complexes and any unattached virions. Then feed the cells with 100 microliters per well of cell culture medium supplemented with 10%fetal bovine serum.
Then return the cells to the cell culture incubator for 48 hours. Two days after the infection, wash the cells two times with 100 microliters of sterile PBS per well before adding 250 microliters of cell lysis buffer with 10%beta mercaptoethanol to each well. Pipette up and down at least five times with scratching to speed up the lysis process and transfer the cell lysates to sterile labeled 1.5 milliliter tubes.
Add an equal volume of 70%ethanol to each tube and pipette the lysate solution up and down four to five times. When the lysate suspensions are clear, transfer each solution into labeled silica-based columns in two milliliter collection tubes for centrifugation. Discard the flow-through keeping the columns in the same collection tubes and add 700 microliters of Wash Buffer 1 to each column for a second centrifugation.
After discarding the flow-through, rinse the columns two more times with 500 microliters of Wash Buffer two per wash. After the second wash, transfer the columns to new two milliliter collection tubes for another centrifugation. Add 30 microliters of 42 degree Celsius RNase-Free water to the center of each column for centrifugation and recover the eluted RNA.
For quantitative real-time polymerase chain reaction or QRTPCR analysis add one microliter of gene specific forward and reverse primers from 10 micromolar stocks along with 0.25 microliters of reverse transcriptase mix per reaction. Note that primers which recognize Zika virus genetic material will detect viral genes and allow for infection quantification. Next, add 12.5 microliters of SYBR Green Mix followed by 25 microliters of water to each reaction adding 15 microliters of Master Mix in each well of 96-well QPCR plate.
When using a Master Mix, add 100 nanograms of the RNA sample to the mix and pipette the Master Mix into individual wells of the QRTPCR plate. Note that samples should be run in triplicate on the same QRTPCR plate. Then run the samples on a quantitative PCR machine according to the parameters outlined in the table.
Clicking the melt curve tab in the system software to monitor the melt curve. The melt curve will show single peak in all of the samples for a particular gene to confirm the presence of only one amplicon. For optimum results, use 0.5 degree Celsius temperature increments between steps and a minimum holding time of 10 seconds in the melt curve protocol.
Then click the quantification data tab to obtain a quantitative cycle for each sample and export the data to a spreadsheet for further quantification analysis. The human serum samples can be categorized into three different groups:dengue virus infection confirmed samples, dengue virus antibody confirmed samples, and healthy sera with no dengue virus neutralizing antibodies or RNA. After 48 hours of infection, QRTPCR analysis demonstrates that most sera containing dengue virus serotype one to four antibodies are able to enhance Zika virus replication at different levels.
The highest increase in Zika virus titres is found in macrophages treated with sera-containing dengue virus serotype two and four antibodies compared to serotype one and three which show a relatively lower induction of Zika virus. It is important to maintain a sterile environment, to wear the proper personal protection equipment, and to always keep the thawed virus serum samples and QPCR reagents on ice. This protocol can easily be modified to study antibody-dependent enhancement of other flavivirus such as yellow fever virus, dengue virus, or West Nile virus using a panel of patient serum.
This technique will be highly useful for carrying out future antibody-dependent enhancement studies in the arbovirus or virology fields. Proper biosafety level two personal protection equipment should be used at all times and all viral waste should be decontaminated in 10%bleach for 30 minutes before disposal.
