Virus Propagation and Cell-Based Colorimetric Quantification

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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 ....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

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.