Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Summary

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Abstract

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-α (IFN-α), IFN-β and IFN-γ as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Introduction

Zika Virus (ZIKV) is an important human pathogen associated with microcephaly and poor pregnancy outcomes^1,4. ZIKV belongs to the set of medically relevant flaviviruses that can cause neurological defects such as the Dengue, West Nile, and St. Louis encephalitis viruses. The main mode of viral transmission is by the mosquito vector Aedes aegypti, and, in addition, sexual transmission has also been reported^5,6. ZIKV has become a major global health issue due to the expanding geographical distribution of the mosquito vector and its strong correlation with birth defects. ZIKV was first isolated in 1947 from a sentinel rhesus monkey in the Z....

Protocol

Note: A schematic outline of the work flow is presented in Figure 1.

1. Cells

1. Use Vero cells for Zika virus production and analysis of viral replication cycle.
2. Prepare complete growth media containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10 mM HEPES.
3. Culture Vero cells with the specified complete growth medium at 37 °C with 5% CO₂.

2. Zika Virus Production

1. Harvest the Vero cells at 80% cell density using 0.25% trypsin in T-75 flask and count the cells.
   1. A....

Results

A Zika viral strain (PRVABC59; GenBank accession number KU501215) of the Asian genotype was utilized in this study¹². Vero cells at 80% confluency were used for investigating de novo Zika viral infection. For viral production and subsequent virological characterization, an early passage (P3) Zika virus was employed. The viral plaques were observed on the second day of infection. Zika viral progenies released from the initially infected cell can spread to neighboring ce.......

Discussion

Here, a streamlined protocol for culturing Zika virus in vitro is presented. Critical steps including, identifying optimum end points for expanding virus culture, measuring titer, and quantifying genome replication were provided. Zika virus is a human pathogen, so, while handling infectious agents, biosafety procedures are to be strictly followed. A monkey kidney cell line, Vero, was used for demonstrating various virological assays. Zika viral replication kinetics may differ in cells of various tissues and spec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s modified Eagle’s medium (DMEM)	Sigma Life Science	D5796
HEPES	Life Technologies	15630080
Glutamax	Life Technologies	35050061
2.5% Trypsin, 10x [-] Phenol Red	Corning	25-054-C1
Trypan Blue Stain 0.4%	Life Technologies	T10282
Countess – Automated Cell Counter	ThermoFisher Scientific	C10227
Countess-cell counting  chamber slides	ThermoFisher Scientific	C10283
Rneasy Mini Kit	Qiagen	74104
Nanodrop 2000	Thermo Scientific	Nanodrop 2000
mouse monoclonal anti-dsRNA antibody J2	English & Scientific Consulting Kft.	10010200
Goat anti-rabbit IgG Alexa Fluor 594	Life Technologies	A11020
SUPERSCRIPT III RT	Life Technologies	18080085
SYBR QPCR SUPERMIX W/ROX	Life Technologies	11744500
QuantStudio12K Flex Real-Time PCR System	Thermo Fischer	4471088
RNase-Free DNase	Promega	M6101
Vero Cell Line	ATCC	CCL-81
Zika viral strain PRVABC59	Centers for Disease Control and Prevention (CDC)
IL-6	Peprotech	200-06
IL-1 alpha	Peprotech	200-01A
TNF-alpha	Peprotech	300-01A
Interferon alpha A	R & D Systems	11100-1
Interferon beta	Peprotech	300-02BC
Interferon gamma	Peprotech	300-02
Centrifuge 5415R	Eppendorf	5415R
Centrifuge 5810R	Eppendorf	5810R
Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI	Nikon	Visit Nikon for Request