Virus Propagation and Cell-Based Colorimetric Quantification

Podsumowanie

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Streszczenie

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barré syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

Wprowadzenie

Zika virus (ZIKV) infection is an emerging mosquito-borne viral disease. The first isolation of ZIKV was in Uganda in 1947^1,2; it remained neglected from 1947 to 2007, as the clinical symptoms are most commonly asymptomatic and characterized by self-limiting febrile illness. In 2007, the Zika epidemic began in the Yap islands^3,4, followed by larger epidemics in the Pacific regions (French Polynesia, Easter Island, Cook Islands, and New Caledonia) from 2013 to 2014^5,6,7,8, where the severe neurological complication Guillain-Barré syndrome (GBS) was reported in adults for the first time⁹. During 2015 and 2016, the first widespread ZIKV epidemic swept across the Americas after the emergence of the Asian genotype of ZIKV in Brazil in as early as 2013¹⁰. During this outbreak, 440,000 to 1.3 million cases of microcephaly, and other neurological disorders, were reported in newborn babies¹¹. There is currently no specific cure or treatment for ZIKV infection; hence, there is an urgent medical need for ZIKV vaccines capable of preventing infections, particularly during pregnancy.

Virus quantification is a process to determine the number of viruses present in a sample. It plays an important role in research, and academic laboratories involve many fields, such as medicine and life sciences. This process is also important in commercial sectors, such as the production of viral vaccines, recombinant proteins, viral antigens, or antiviral agents. Many methods or assays can be used for virus quantification¹². The choice of methods or assays normally depends on the virus characteristics, desired level of accuracy, and the nature of the experiment or research. In general, methods of quantifying viruses can be divided into two categories: molecular assays that detect the presence of viral nucleic acid (DNA or RNA) and assays that measure virus infectivity in vitro¹². Quantitative polymerase chain reaction (qPCR, for DNA) or quantitative reverse transcription polymerase chain reaction (qRT-PCR, for RNA)¹³ and digital droplet PCR¹⁴ are examples of common molecular techniques used to quantitate the viral nucleic acid in a given sample¹⁵. However, these highly sensitive molecular techniques cannot differentiate between viable and non-viable viruses¹⁵. Therefore, research that requires information on biological features, such as virus infectivity on cells, cannot be completed using the abovementioned molecular techniques; assays that can measure and determine the presence of viable viruses are needed. Assays that measure virus infectivity include the plaque forming assay (PFA), 50% tissue culture infectious dose (TCID50), the fluorescent focus assay, and transmission electron microscopy (TEM)¹².

The PFA, developed by Renato Dulbecco in 1952, is one of the most commonly used methods for virus titration, including for ZIKV¹⁶. It is used to directly determine the viral concentrations for infectious lytic virions. The method is based on the ability of a lytic virus to produce cytopathic effects (CPEs; zones of cell death or plaques, an area of infection surrounded by uninfected cells) in an inoculated cell monolayer after viral infection. However, there are several drawbacks to the assay that affect its utility. The assay is time-consuming (takes approximately 7-10 days, depending on viruses), CPE-dependent, and prone to errors. In the present study, we report an immunocolorimetric technique, the focus forming assay (FFA), for detecting and quantifying ZIKV in 24-well plate and 96-well plate formats.

Protokół

1. Virus propagation

1. Cell preparation
   1. Grow Vero cells in a 75 cm² cell culture flask containing 12 mL of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (see Table of Materials). Incubate the cells in a cell culture incubator at 37 °C with 5% CO₂.
   2. Monitor the cells under a microscope; once the cells reach 70%-90% confluency, they are ready to be used (Figure 1A) .
      NOTE: The Vero cells double approximately every 24 h¹⁷. Dilutions of 1:10 should take 3 to 4 days to reach 80%-90% confluency in a 75 cm² flask. Monitor the cells daily or every other day.
2. Infection of the cell monolayer
   1. Remove the growth medium from the cell culture flask using a 10 mL serological pipette. Rinse the flask with 3 mL of Dulbecco's phosphate buffered saline (dPBS) 2x using a 5 mL serological pipette.
      NOTE: A beaker with 10% sodium hypochlorite must be prepared to disinfect any discarded infectious liquid waste from flasks/plates.
   2. Add 2 mL of serum-free DMEM (DMEM supplemented with 2 mM L-glutamine without FBS) and 20 µL of ZIKV inoculum into the cell culture flask. Incubate the flask with gentle rocking for 1 h at room temperature to allow virus adsorption.
      NOTE: Titration of the initial ZIKV stock will be helpful to determine the volume of ZIKV inoculum addition (step 2).
      CAUTION: ZIKV is classified as a biosafety level 2 (BSL-2) pathogen. All procedures with materials containing ZIKV must be conducted in a certified biosafety cabinet and follow all the laboratory standard operating procedures (SOPs) with appropriate personal protective equipment.
3. Addition of the overlay medium
   1. After the 1 h incubation, remove and discard the diluted virus inoculum using a 5 mL serological pipette. Rinse the cell culture flask with 3 mL of dPBS 2x.
   2. Add 12 mL of maintenance media (DMEM supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin) into the cell culture flask to maintain the infected cells.
   3. Incubate the infected Vero cells for 3 days in a cell culture incubator at 37 °C with 5% CO₂.
      NOTE: Monitor daily for the CPE of infected Vero cells under a microscope.
4. Harvesting of cell culture supernatant containing ZIKV
   1. On day 3 post-infection, cell rounding and detachment (CPE) can be observed (Figure 1B-D). Harvest the cell culture supernatant containing the ZIKV into a 50 mL centrifuge tube using a 10 mL serological pipette.
   2. Use a 0.22 µm Polyethersulfone syringe filter (see Table of Materials) to filter the cell culture supernatant. Aliquot 500 µL of the filtered cell culture supernatant into 2.0 mL screw cap tubes and store at -80 °C until further use.
      NOTE: Harvested ZIKV can be stored at -80 °C for long-term storage.
      ​CAUTION: Extra precautions must be taken if a syringe needle is used, as the needle can puncture the skin. Laboratory SOPs need to be established.

2. Virus quantification

1. Cell preparation
   1. Seed Vero cells in designated plates (24-well plate: 0.5 mL/well, 7.5 x 10⁴ cells/well; 96-well plate: 0.1 mL/well, 1.5 x 10⁴ cells/well) and incubate the plate overnight in a cell culture incubator at 37 °C with 5% CO₂.
      NOTE: There are five different time points to be tested (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection); therefore, prepare one plate for each time point.
   2. After the 24 h incubation and once the cells reach 70%-90% confluency (Figure 1E), the plate is ready for infection. Proceed to prepare the diluted virus stock (step 2.2) prior to infection of the cell monolayer.
2. Virus dilution
   1. For 24- and 96-well plates, prepare six sterile 1.5 mL microcentrifuge tubes for each plate to carry out a tenfold dilution (10^-1 up to 10^-5), including a negative control. For the experiment setup for a 24-well plate, add 450 µL of serum-free DMEM into all six microcentrifuge tubes. For the experiment setup for a 96-well plate, add 135 µL of serum-free DMEM into six tubes.
      NOTE: Label each tube clearly to indicate the dilution of its contents before performing the tenfold serial dilution.
   2. To perform serial dilution, for the 24-well plate experiment setup, add 50 µL of ZIKV stock that was harvested from step 1 into the 10^-1 tube that contains 450 µL of serum-free DMEM. For the 96-well plate experiment setup, add 15 µL of ZIKV stock into the 10^-1 tube that contains 135 µL of serum-free DMEM.
   3. Vortex each tube to mix the virus and medium. This is the first tenfold dilution.
      NOTE: Proper mixing of virus and culture medium in each dilution tube is required to ensure accurate serial dilution.
   4. For the 24-well plate experiment setup, use a fresh pipette tip to resuspend the 10^-1 tube and transfer 50 µL of diluted ZIKV into the 10^-2 tube as a second tenfold dilution. For the 96-well plate experiment setup, use a fresh pipette tip to resuspend the 10^-1 tube and transfer 15 µL of diluted ZIKV into the 10^-2 tube as a second tenfold dilution.
   5. Repeat step 2.2.4 four times until the fifth tube (exclude negative control).
      NOTE: Use a fresh pipette tip after each pipetting step to avoid cross-contamination.
3. Infection of the cell monolayer
   1. Remove and discard the conditioned medium from each well (24-well plate: 0.5 mL/well; 96-well plate: 0.1 mL/well).
   2. Rinse each well with dPBS 2x (24-well plate: 300 µL/well; 96-well plate: 60 µL/well).
   3. Working from the highest to the lowest dilution, add each serially diluted virus inoculum into the well (24-well plate: 200 µL/well; 96-well plate: 40 µL/well).
      ​NOTE: The infection of each dilution will be performed in duplicated wells. Maintain two uninfected wells as the negative control. Label the plate and wells clearly to avoid confusion. Change the pipette tips after each pipetting step to prevent cross-contamination.
   4. Incubate the plate with gentle rocking for 1 h at room temperature to allow virus adsorption.
4. Addition of the overlay cell culture medium
   1. After 1 h of incubation, remove and discard the virus suspension from the lowest concentration to the highest.
   2. Wash the infected cells with dPBS 2x (24-well plate: 300 µL/well; 96-well plate: 60 µL/well).
   3. Overlay the well with DMEM (supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin) and 1.5% low viscosity carboxymethyl cellulose (CMC; see Table of Materials) (24-well plate: 1 mL/well; 96-well plate: 0.2 mL/well).
   4. Incubate the plate in a cell culture incubator at 37 °C with 5% CO₂.
      NOTE: Do not disturb the plate during this incubation period.
   5. Take the plate out from the cell culture incubator for staining at different time points (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection).

3. Staining

1. Cell fixation
   1. After the specific hours of incubation (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection), remove and discard the overlay medium and wash the cells 3x with 1x PBS. For the 96-well plate, remove and discard the overlay medium and wash the cells 3x with 60 µL/well of 1x PBS with a multichannel pipette.
   2. Add 4% paraformaldehyde to fix the cells (24-well plate: 300 µL/well; 96-well plate: 60 µL/well). Incubate the plate at room temperature for 20 min.
      CAUTION: Paraformaldehyde is a solid polymerized formaldehyde. It is a flammable, solid, white crystalline powder that releases formaldehyde gas when mixed with water or heated. All procedures involving the use of paraformaldehyde must be conducted in a certified chemical fume hood or approved exhausted enclosures following laboratory SOPs specific to formaldehyde-containing chemicals.
   3. After 20 min, discard the paraformaldehyde and wash the cells 3x with 1x PBS. Perform permeabilization, blocking, and antibody incubations following steps 3.2-3.5.
      NOTE: When discarding 4% paraformaldehyde, follow the university or institute's waste disposal procedure.
2. Cell permeabilization
   1. Add 1% octylphenoxy poly(ethyleneoxy)ethanol (see Table of Materials) to permeabilize the cells (24-well plate: 200 µL/well; 96-well plate: 40 µL/well). Incubate the plate at room temperature for 15 min.
   2. After 15 min, discard the octylphenoxy poly(ethyleneoxy)ethanol and wash the cells 3x with 1x PBS.
3. Blocking
   1. Add 3% skim milk to reduce the nonspecific binding in the sample (24-well plate: 500 µL/well; 96-well plate: 100 µL/well). Incubate the plate at room temperature for 2 h.
   2. After 2 h, discard the 3% skim milk and wash the cells 3x with 1x PBS.
      ​NOTE: This is a stopping point. After adding 3% skim milk, the plates can be stored at 4 °C up to 2 days before primary antibody insertion.
4. Primary antibody incubation
   1. Add anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15; primary antibody, see Table of Materials) diluted in 1% skim milk (1:1,000 ratio) (24-well plate: 200 µL/well; 96-well plate: 40 µL/well). Incubate the plate at 37 °C for 1 h.
   2. After 1 h, remove the solution containing the monoclonal antibody and wash the cells 3x with 1x PBS.
5. Secondary antibody incubation
   1. Add goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP) (see Table of Materials) diluted in 1% skim milk (1:1,000 ratio) (24-well plate: 200 µL/well; 96-well plate: 40 µL/well). Incubate the plates at 37 °C for 1 h.
   2. After 1 h, remove the solution containing the secondary antibody and wash the cells 3x with 1x PBS.
6. Substrate incubation
   1. Add 3,3'diaminobenzidine (DAB) peroxidase substrate (see Table of Materials) and incubate the plate for 30 min in the dark (24-well plate: 200 µL/well; 96-well plate: 40 µL/well). After 30 min, stop the reaction by washing the wells with water.
   2. Air-dry the plates overnight and proceed to foci enumeration after the plates are completely dry.

4. Determination of the virus titer

1. Manual foci counting process (24-well format)
   1. Foci can be visualized under a stereo microscope. Select the dilution that produces 50-200 foci.
   2. Count the foci for each replicate of the selected dilution. Calculate the average number of foci for each of the selected dilutions.
   3. Calculate the foci forming unit per mL (FFU/mL) for each sample with the formula below: FFU/mL = average number of foci counted/(dilution x volume of diluted virus added).
2. Automated foci counting process (96-well format)
   1. Scan the 96-well plate on a commercial software analyzer.
      1. Double click on the software (see Table of Materials) to open.
      2. Click on Switch Suites and ensure that the suite is switched to any one that is applicable (Supplementary Figure 1A). Click OK.
      3. Begin the scan by clicking Scan > Full plate scan (Supplementary Figure 1B).
      4. Click on Dashboard to ensure the capture format is the 96-well format (Supplementary Figure 2A).
      5. Select the centering mode as "Auto Prealignment User Verified" (Supplementary Figure 2B).
      6. Click on Eject to load the plate.
         NOTE: Open the lid of the plate and place it upward with Row A at the top (Supplementary Figure 3A).
      7. Enter the plate name and click on OK. Click on Load to load the plate.
      8. Click on Start to start the scan. After that, calibrate the positions for A1, A12, and H1 using the buttons "Up, Down, Left, Right" and click on Confirm (Supplementary Figure 3B).
         NOTE: The software will start scanning each well.
      9. Once the scanning is complete, an overview image will pop up. Click on Close.
      10. Exit the scan by clicking on Back to switchboard.
   2. Count the 96-well plate using the software.
      1. Click Count > Smart Count.
      2. On the software, it will show "Step 1 of 5: Select plates to count". Click on Load plate(s). Tick the whole folder and click on Select to import the scanned plate (Supplementary Figure 4A).
      3. On the software, it will show "Step 2 of 5: Define counting parameters". Click on Adjust parameters, and the parameters will be displayed (diffuse process; small/normal/large/largest/largest +1; spot separation; counted area; background balance; fiber removal; gating).
         NOTE: Start with one well, and check back and forth to find the best settings for all the wells.
      4. Once done, click on Next (Supplementary Figure 4B).
      5. On the software, it will show "Step 3 of 5: Select/unselect wells". Once the selection of the well to be counted is complete, click on Next to proceed.
         NOTE: The wells selected to be counted will appear with a "C" at the top right corner of each well (Supplementary Figure 5A).
      6. On the software, it will show "Step 4 of 5: Output Settings". Click on View/Modify Output Settings to check if the settings (such as image format) are suitable. Click on Save and Exit > Next (Supplementary Figure 5B).
      7. On the software, it will show "Step 5 of 5: Start AutoCount". Click on Start AutoCount (Supplementary Figure 6A).
      8. Once complete, click on Exit AutoCount.
3. Perform quality control (QC) in the commercial software analyzer.
   1. On the switchboard page, click on Quality Control > Add plate(s). Tick the whole folder to import the counted plate, click on Select > Start QC (Supplementary Figure 6B).
   2. Double-click on each well to audit the spots. Click on Count to remove any spots. The software will start counting.
      NOTE: Ensure that "Spots: Remove" is ticked. Completion of the counting can be indicated by the counted number of foci, for example "30" (Supplementary Figure 7A).
   3. Once the counting ends (indicated by the counted number of foci, for example "30"), click "Yes" to remove spots (Supplementary Figure 7B). Triple left-click on the spot to be removed. Right-click to finish, then click on Yes.
   4. Click on Finish Plate and Go to Project Overview once the QC is complete.
4. Perform imaging using the software analyzer.
   1. Check the scanned, counted, and QC plate image (Figure 2).

Wyniki

ZIKV can be quantified using the FFA, as outlined schematically in Figure 3. For the 24-well plate, the infected Vero cells were fixed at 48 h, 60 h, 72 h, 84 h, and 96 h post-infection. The results showed that the cells remained intact (no cell detachment was observed) after 96 h (4 days) post-infection (Figure 4 and Supplementary Figure 8A-E). The appearance of virus foci was first observed at 48 h (2 days) post-infection (

Dyskusje

There are several assays to determine virus titer; the PFA has a similar virus quantitation protocol as the FFA, in which the virus inoculum is diluted to allow individual plaques or foci to be distinguished. After staining, each plaque or foci indicates a single infectious particle in the inoculum¹⁹. The PFA is stained with crystal violet to visualize plaque formation caused by cell lysis or death. Hence, the PFA is more time-consuming, as it requires a longer time for the virus to cause CPEs, an...

Materiały

Name	Company	Catalog Number	Comments
0.22 µm Polyethersulfone syringe filter	Sartorius	S6534-FMOSK
1.5 mL microcentrifuge tube	Nest	615601
10 mL sterile serological pipette	Labserv	14955156
1x Dulbecco’s phosphate-buffered saline (dPBS)	Gibco	14190-136
2.0 mL Screw cap tube	Axygen	SCT-200-SS-C-S
24-well plate	Corning	3526
25 mL Sterile serological pipettes	Labserv	14955157
3,3'Diaminobenzidine (DAB) peroxidase substrate	Thermo Scientific	34065
37 °C incubator with 5% CO2	Sanyo	MCO-18AIC
5 mL sterile serological pipette	Labserv	14955155
50 mL centrifuge tube	Falcon	LAB352070
75 cm2 tissue culture flask	Corning	430725U
96-well plate	Falcon	353072
Anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15)	MilliporeSigma	MAB10216
Autoclaved 20x Phosphate buffered saline (PBS)	N/A	N/A	22.8 g of 8 mM Na2HPO4, 4.0 g of 1.5 mM KH2PO4, 160 g of 0.14 M NaCl, 4.0 g of 2.7 mM KCl, 1 L of MilliQ H2O
Biological safety cabinet, Class II	Holten	HB2448
CTL S6 Universal ELISpot/FluoroSpot Analyzer	ImmunoSpot, Cellular Technology Limited (CTL)	CTL-S6UNV12	Commercial software analyzer
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	12800-017
Fetal bovine serum (FBS)	Bovogen	SFBS
Goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP)	MilliporeSigma	12-349
Hemacytometer	Laboroptik LTD	Neubauer improved
IGEPAL CA-630 detergent	Sigma-Aldrich	I8896	Octylphenoxy poly(ethyleneoxy)ethanolIGEPAL
Inverted microscope	ZEISS	TELAVAL 31
Laboratory rocker	FINEPCR	CR300
L-Glutamine	Gibco	25030-081
Low viscosity carboxymethyl cellulose (CMC)	Sigma-Aldrich	C5678
Multichannel micropipette (10 - 100 µL)	Eppendorf	3125000036
Multichannel micropipette (30 - 300 µL)	Eppendorf	3125000052
Paraformaldehyde	Sigma-Aldrich	P6148
Penicillin-streptomycin	Gibco	15140-122
Single channel pipettes (10 - 100 µL)	Eppendorf	3123000047
Single channel pipettes (100 - 1000 µL)	Eppendorf	3123000063
Single channel pipettes (20 - 200 µL)	Eppendorf	3123000055
Skim milk	Sunlac Low Fat	N/A	Prepare 3% Skim milk in 1x PBS for blocking stage in staining
Sodium Hypochlorite	Clorox	N/A	To disinfect any discarded infectious liquid waste from flasks/plates
Stereomicroscope	Nikon	SMZ1000
Syringe disposable, Luer Lock, 10 mL with 21 G Needle	Terumo	SS10L21G
Vero African green monkey kidney cells	-	ECACC 88020401	Received from collaborator. However, Vero cells obtained from other suppliers should be able to be used with some optimization.