Abstract
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.
Materials
| 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. |
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