Virusformering og cellebaseret kolorimetrisk kvantificering

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

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.

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 2014^5,6,7,8, 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.

Protocol

1. Virus propagation Cell preparation 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. Monitor the cells under a microscope; once the cells reach 70%-90% confluency, they are ready to be used (<stron…

Representative 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 (<strong class="xfig"…

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

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.