Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

Serum samples used in this study were obtained from human participants of a cohort from Columbia. Sample collection was approved by the internal review board (IRB) at Universidad de Pamplona (Columbia, South America) and Los Potios Hospital⁸. The samples were anonymously provided and investigators had no access to patient information. The serum samples were checked for the DENV serotype. The samples were further confirmed to neutralize DENV infection in vitro. For control, serum samples from healthy individuals (HC) from the USA were used.

NOTE: This protocol can be used to examine the ADE of ZIKV replication in any human cell type expressing the Fcγ receptor. The protocol consists of three parts (Figure 1).

1. Cell Seeding and Infection Setup

NOTE: For this particular study, human primary macrophages or U937 myelomonocytic cell line (ATCC-CRL-1593.2) were used. The cells were maintained in RPMI growth medium supplemented with 10% fetal bovine serum (FBS) at a 37 °C incubator with 5% carbon dioxide (CO₂). All the steps were carried out in a biosafety level 2 (BSL-2) biosafety cabinet in sterile conditions.

1. Seed 3 x 10⁴ cells per well in a sterile flat-bottom 96-well plate along with 100 µL of medium and place them in the 37 °C incubator with 5% CO₂.
2. After seeding the cells, thaw serum samples at room temperature and make 10-fold serial dilutions (i.e., 1/10, 1/100, 1/1,000, 1/10,000) by mixing the samples in serum-free medium. Aliquot the dilutions into a sterile 96-well plate. Use the virus without serum as an additional control.
3. Thaw the ZIKV stock at 37 °C in a water bath for 1 – 2 min and quickly transfer them to ice for future use.
4. Add a 0.1 multiplicity of infection (MOI) equivalent amount of ZIKV strain MR766 to the serum aliquots.
   NOTE: Make sure that the dilution factor remains constant and the total volume is ~200 µL; that is enough for triplicates of each treatment. Keep three wells uninfected to use as negative control for downstream analysis.
5. Incubate the serum dilutions with the added virus in the 37 °C incubator with 5% CO₂ for 1 h in order to allow the DENV antibodies to form complexes with the Zika virions (for convenience, hereafter referred to as "immune complexes").
6. Aspirate the media from the cells that were seeded in the flat-bottom 96-well plate. Wash the cells with sterile 1x phosphate-buffered saline (1x PBS).
7. Add 50 µL of the immune complexes to each well and incubate them in the 37 °C incubator with 5% CO₂ for 2 h.
8. After 2 h of incubation, aspirate the media containing the immune complexes from the cells, using a multichannel pipette, and wash the cells 2x with 1x PBS to completely remove the immune complexes and any unattached virions.
9. Add 100 µL of fresh complete media supplemented with 10% FBS to each well and incubate the cells in the 37 °C incubator with 5% CO₂ for 48 h.

2. RNA Extraction

1. Remove the 96-well plate from the incubator and transfer it to the biosafety cabinet.
2. Aspirate the media, wash the cells 2x with 1x PBS, and proceed to RNA extraction.
   NOTE: RNA can be extracted by any method of choice (refer to the Table of Materials).
3. Add 250 µL of cell lysis buffer with 10% β-mercaptoethanol per well. Pipette the buffer up and down at least 5x along with scratching the cells with the pipette tip to speed up the lysis procedure.
4. Transfer the cell lysates to new, labeled, sterile 1.5 mL tubes.
5. Add an equal amount of 70% ethanol (250 µL). Pipette up and down 4x – 5x until the mixture is clear.
6. Transfer the mixture (~500 µL) to labeled silica-based columns in 2 mL collection tubes and centrifuge at 15,000 x g for 30 s. Discard the flow through and keep the columns in the same collection tubes.
7. Add 700 µL of wash buffer 1 to each column and centrifuge at 15,000 x g for 30 s. Discard the flow through and keep the columns in the same collection tubes.
8. Add 500 µL of wash buffer 2 to each column and centrifuge at 15,000 x g for 30 s. Discard the supernatant. Repeat this step 2x.
9. Transfer the columns to new 2 mL collection tubes and centrifuge at 15,000 x g for 2 min. Ensure that silica-based columns get completely dry and there is no ethanol left from wash buffer 2.
10. Discard the 2 mL tubes and place the columns in new, sterile, labeled, 1.5 mL recovery tubes.
11. Add prewarmed (42 °C) 30 µL of RNase-free water at the center of each column and centrifuge at 15,000 x g for 1 min.
12. Recover the eluted RNA and quantify the samples, using a spectrophotometer at a 260 nm wavelength.
    NOTE: Ideally, determine the purity of the RNA by calculating the ratio between the absorbance values at 260 nm and 280 nm. Purified RNA's 260/280 ratio is ideally between 1.8 – 2.0.

3. Quantitative Real-time Polymerase Chain Reaction

NOTE: Quantitative real-time polymerase chain reaction (qRT-PCR) can be carried out by using any SYBR green mix which usually is composed of SYBR Green I dye, Taq DNA polymerase, deoxynucleotide triphosphates (dNTPs), and a passive dye. Any qPCR machine capable of the detection of SYBR green can be used to perform the reaction and acquire the data. For this experiment, a one-step RT-PCR kit was used which had a cocktail of SYBR Green I, ROX dye, Taq DNA polymerase, dNTPs, and an additional mixture of reverse transcriptase (refer to Table of Materials). The primers designed against the envelope region and used in this study to quantify ZIKV genomic levels are CCGCTGCCCAACACAAG for ZIKV-qF and CCACTAACGTTCTTTTGCAGACAT for ZIKV-qR. As a control, human β-2-microglobulin (B2M) was measured and used to normalize the expression of ZIKV (housekeeping gene). The primer sequences to quantify the B2M gene expression used in this study are CTCCGTGGCCTTAGCTGTG for B2M-qF and TTTGGAGTACGCTGGATAGCCT for B2M-qR. Ensure to put three technical replicates for each sample for both the ZIKV and the B2M genes. The setup of the RT-PCR is briefly described below.

1. RT-PCR setup
   1. Use 100 ng of RNA per sample.
   2. Add 1 µL from 10 µM stocks of both forward and reverse primers of a particular gene for each reaction.
   3. Add 0.25 µL of reverse transcriptase mix per sample.
   4. For each individual reaction, add 12.5 µL of SYBR Green mix.
   5. Add up to 25 µL of water. Make a master mix of all the above-mentioned reagents except RNA, aliquot it in the 96-well PCR plate, and add RNA last.
   6. Seal the plate with transparent adhesive tape.
   7. Centrifuge the plate at 1,000 x g for 1 min to mix the reagents.
   8. Put the plate in the qPCR machine.
2. RT-PCR profile
   NOTE: Use the RT-PCR profile shown in Table 1.
   1. Incubate the samples at 50 °C for 10 min in order to ensure the synthesis of complementary DNA (cDNA).
   2. After the cDNA synthesis, activate the Taq DNA polymerase at 95 °C for 5 min.
   3. Perform 40 cycles of denaturation at 95 °C for 10 s and primer annealing and extension at 60 °C for 30 s.
   4. Put the additional melt curve step (65°C) in the end.
3. Data analysis
   1. Monitor the melt curve by clicking the melt curve tab in the program used to run the qPCR machine, which, ideally, shows a single peak in all the samples for a particular gene to confirm the presence of only one amplicon and an amplification value more than 1.6 if the algorithm considers 2 as the 100% amplification value (the amplification value varies according to the algorithm used by the qPCR machine). Make sure to use 0.5 °C temperature increments between steps and a minimum holding time of 10 s in the melt curve protocol, for optimal results.
   2. After making sure there are a single amplicon and good amplification value, first click on the quantification data tab to get a quantitative cycle (Ct/Cq value) of each sample and export to Microsoft Excel.
   3. Using a spreadsheet, calculate the average of each sample using the Ct value. Next, click in the formulas and select the average. The average CT values of each sample (replicate) are used for further analysis.
   4. Next, calculate the ΔCt using the formula ΔCt = average Ct of the target gene – average Ct of the control gene (in this case, ΔCt = average Ct of the ZIKV gene – average Ct of the B2M gene).
   5. Calculate ΔΔCt to normalize the data (in this case, ΔΔCt = ΔCt ZIKV samples – B2M samples).
   6. Calculate the fold increase gene expression by typing the formula 2^-(ΔΔCt) for every single ΔΔCt normalized value. The results of the fold increase to perform statistical tests can be obtained as described in earlier studies^9,10.