This protocol enables identifying and quantifying the Zika virus without relying on cytopathic effect. It depends on antibody recognition of virus component. The use of virus-specific antibody can help in identifying different virus serotypes in mixed populations.
This method has advantages over classical plaque-forming assays. It's faster and enabled for high-throughput applications when applying with an automated imaging system for fast cell counting. The proposed protocol is highly adaptable.
With appropriate modifications, the proposed protocol can be applied to various cell types and viral targets. Start by growing Vero cells in a 75-square=centimeter cell culture flask that contains 12 milliliters of DMEM supplemented with 10%FBS and two millimoles of L-glutamine. Place the flask in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.
To infect the cell monolayer, use a 10-milliliter serological pipette to remove the growth medium from the cell culture flask. Using a five-milliliter serological pipette, rinse the flask with three milliliters of DPBS twice. Next, add two milliliters of serum-free DMEM and 20 microliters of Zika virus inoculum into the cell culture flask.
Let the flask incubate at room temperature for one hour with gentle rocking to encourage virus adsorption. At the end of the incubation, using a five-milliliter serological pipette, carefully remove and discard the diluted virus inoculum from the cell culture flask. Rinse the cell culture flask twice with three milliliters of DPBS.
Then, add 12 milliliters of maintenance media into the cell culture flask to maintain the infected cells. Incubate the infected Vero cells for three days in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide. After three days of incubation, use a 10-milliliter serological pipette to harvest the cell culture supernatant containing the Zika virus into a 50-milliliter centrifuge tube.
For virus quantification, seed Vero cells in designated plates and allow them to grow overnight at 37 degrees Celsius with a 5%carbon dioxide atmosphere. Prepare six sterile 1.5-milliliter microcentrifuge tubes for each plate to perform a ten-fold serial dilution, including an extra tube for a negative control. For a 24-well plate setup, add 450 microliters of serum-free DMEM to all six microcentrifuge tubes.
For a 96-well plate set up, dispense 135 microliters of serum-free DMEM to six additional tubes. To perform serial dilution for the 24-well plate experiment, add 50 microliters of the Zika virus stock into the 10 to minus one tube containing 450 microliters of serum-free DMEM. For the 96-well plate experiment, add 15 microliters of Zika virus stock into the 10 to minus one tube containing 135 microliters of serum-free DMEM.
Vortex each tube to thoroughly mix the virus and medium, ensuring an even distribution of virus particles within the dilution. Using a fresh pipette tip, resuspend the 10 to minus one tube and transfer 50 and 15 microliters of diluted Zika virus into the 10 to minus two tube as a second ten-fold dilution for the 24-and 96-well plates, respectively. Remove and discard the condition medium for each well of the appropriate plate.
Rinse each well with DPBS twice to remove any residues. Starting with the highest dilution, add the serially diluted virus inoculum into the wells, working towards the lowest dilution. After one hour of incubation, remove and discard the virus suspension from the wells, starting with the lowest to the highest concentration.
Wash the infected cells with DPBS twice to remove any traces of virus suspension. Overlay the well with DMEM and 1.5%low-viscosity carboxymethylcellulose. Incubate the plate in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide.
After incubation, remove and discard the overlay medium and wash the cells three times with PBS. For the 96-well plate, use a multichannel pipette to remove and discard the overlay medium, and wash the cells three times with 60 microliters of PBS per well. Add 4%paraformaldehyde to fix the cells and incubate the plate at room temperature for 20 minutes.
After 20 minutes, discard the paraformaldehyde and wash the cells thrice with PBS. Next, add 3, 3'diaminobenzidine peroxidase substrate and incubate the plate for 30 minutes in the dark. After 30 minutes, stop the reaction by washing the wells with water.
Air-dry the plates overnight and proceed to foci enumeration. Count the foci for each replicate of the selected dilution and calculate the average number of foci for each. The infected Vero cells were fixed at different time points post-infection.
For the 2-well plate, the first appearance of virus foci was observed 48 hours post-infection, although the foci size was too small to count accurately. After 96 hours post-infection, there were no signs of cell detachment. At 60 hours post-infection, the foci size had increased to an optimal level for counting.
Subsequently, as time progressed, the foci became larger and began to merge or overlap with one another in intensity, forming clusters that grew over time. Therefore, foci formed 60 hours after the infection were chosen to determine the Zika virus titre in a 24-well plate. For the 96-well plate, the cells remained intact after 72 hours post-infection.
The appearance of virus foci was first observed 24 hours post-infection. However, up to 36 hours post-infection, the foci size was too small. The optimal foci size was achieved at 48 hours post-infection.
At the latter time points, overlapped or merged foci were observed, and the number of overlapped foci increased over time. The foci formed 48 hours after the infection was chosen to determine the virus titre of Zika virus isolates. Gently add DPBS down the side of each well and rock the plates backward and forward one to three times to remove cellular debris and excess media.
This technique will greatly benefit researchers in Zika research, and can also be broadly adapted to quantify other clinically important viruses, making it a valuable tool for virus surveillance and diagnosis.
