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.
Zikavirus (ZIKV) er en mygbåren virus tilhørende slægten Flavivirus. ZIKV-infektion har været forbundet med medfødte hjerneabnormiteter og potentielt Guillain-Barré syndrom hos voksne. Forskning i ZIKV for at forstå sygdomsmekanismerne er vigtig for at lette vaccine- og behandlingsudvikling. Metoden til kvantificering af vira er afgørende og grundlæggende inden for virologi. Fokusdannende assay (FFA) er et viruskvantificeringsassay, der detekterer det virale antigen med antistoffer og identificerer infektionsfoci af celler ved hjælp af peroxidaseimmunfarvningsteknikken. Den aktuelle undersøgelse beskriver virusudbredelses- og kvantificeringsprotokollen ved hjælp af både 24-brønd og 96-brønd (høj gennemstrømning) formater. Sammenlignet med andre lignende undersøgelser har denne protokol yderligere beskrevet foci størrelsesoptimering, som kan tjene som vejledning til at udvide brugen af dette assay til andre vira. For det første udføres ZIKV-formering i Vero-celler i 3 dage. Den kultursupernatant, der indeholder ZIKV, høstes og kvantificeres ved hjælp af FFA. Kort fortalt podes viruskulturen på Vero-celler og inkuberes i 2-3 dage. Focidannelse bestemmes derefter efter optimerede farvningsprocesser, herunder cellefiksering, permeabilisering, blokering, antistofbinding og inkubation med peroxidasesubstrat. De farvede virusfoci visualiseres ved hjælp af et stereomikroskop (manuel tælling i 24-brøndformat) eller softwareanalysator (automatiseret tælling i 96-brøndformat). FFA giver reproducerbare, relativt hurtige resultater (3-4 dage) og er egnet til brug for forskellige vira, herunder ikke-plakdannende vira. Derefter er denne protokol nyttig til undersøgelse af ZIKV-infektion og kan bruges til at detektere andre klinisk vigtige vira.
Zika virus (ZIKV) infection is an emerging mosquito-borne viral disease. The first isolation of ZIKV was in Uganda in 19471,2; 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 islands3,4, followed by larger epidemics in the Pacific regions (French Polynesia, Easter Island, Cook Islands, and New Caledonia) from 2013 to 20145,6,7,8, 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.
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…
The authors have nothing to disclose.
This research received support from the Ministry of Higher Education Malaysia under the Long-Term Research Grant Scheme (LRGS MRUN Phase 1: LRGS MRUN/F1/01/2018) and funding for the Higher Institution Centre of Excellence (HICoE) program (MO002-2019). Figure 3 in this study that shows the workflow of staining for the foci forming assay is adapted from "DAB Immunohistochemistry" by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates/t-5f3edb2eb20ace00af8faed9-dab-immunohistochemistry.
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. |