Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Virol. Feb 12, 2018; 7(1): 10-20
Published online Feb 12, 2018. doi: 10.5501/wjv.v7.i1.10
Identification of various cell culture models for the study of Zika virus
Kiyoshi Himmelsbach, Eberhard Hildt
Kiyoshi Himmelsbach, Eberhard Hildt, Department of Virology, Paul-Ehrlich-Institut, Langen 63225, Germany
Eberhard Hildt, Center for Infection Research (DZIF), Braunschweig 38124, Germany
Author contributions: Himmelsbach K designed and performed the experiments, analyzed the data and wrote the manuscript; Hildt E coordinated the research, analyzed the data and edited the manuscript.
Supported by the Federal Ministry of Health (BMG) to Hildt E.
Conflict-of-interest statement: There are no conflicts of interest.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Eberhard Hildt, PhD, Professor, Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, Langen 63225, Germany.
Telephone: +49-610-3775411 Fax: +49-610-3771273
Received: October 25, 2017
Peer-review started: October 27, 2017
First decision: November 23, 2017
Revised: December 6, 2017
Accepted: December 13, 2017
Article in press: December 13, 2017
Published online: February 12, 2018
Research background

Zika virus (ZIKV) is an emerging virus transmitted mainly by mosquitos, that has spread during the last decades from Africa, to Asia, over Micronesia to the Americans causing an epidemic in Brazil in the years 2016/2017. In order to propagate the virus in cell culture we investigated various cell lines for their susceptibility to ZIKV infection.

Research motivation

To date ZIKV is mainly propagated in Vero cells derived from kidney epithelial cells from African green monkey. This study aimed to investigate the potential of various cell lines to support the viral life cycle in order to provide researchers with suitable cell culture systems for different issues in the field of ZIKV research.

Research objectives

The objectives of this research were to investigate ten human and non-human cell lines from various tissues (e.g., hepatocytes, keratinocytes and neuronal cells) with regard to their intracellular amount of viral genomes and infectious viral particles upon ZIKV-infection. Moreover, the amount of secreted viral genomes and infectious viral particles was analyzed in the cell culture supernatants. Furthermore, the amount of infected cells was analyzed by immunofluorescence using an Envelope-specific antibody and the amount of NS1 was analyzed by western blot. In order to draw a conclusion whether parts of the innate immune response are responsible for the found differences in viral support, STAT1 distribution and expression was analyzed.

Research methods

Quantification of viral genomes was performed by qPCR. For the detection of genomes from whole cell lysate a standard PCR protocol with SYBR green was used with cDNA as template transcribed from total RNA that was isolated with a Tri-reagent. Viral genomes released into the cell culture supernatant were isolated with a viral RNA isolation kit and subjected to a Taqman-PCR based on a ZIKV-Lightmix Kit. The detection of infectious virus was performed by virus titration assay using serial dilutions from the supernatant or from cleared cellular lysates. The amount of infected cells was analyzed with immunofluorescence microscopy by using an Env-specific antibody and with western blot using NS1-specific antibody. The effect on the innate immunity was monitored by luciferase-reporter assay and STAT1 distribution was analyzed by immunofluorescence microscopy.

Research results

All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines.

Research conclusions

The results presented so far provide a toolbox of cell culture systems for ZIKV research in general. However, the analyzed cells differ strongly with respect to the amount of released viral particles, whereas the amount of genomes amongst the cells in the supernatant and inside of infected cells are more or less equal. This is an important finding, since a lot of research and diagnostic is based on qPCR analysis only.

Research perspectives

Further research should aim on the differences of released viral genomes vs released infectious virus. Are there differences in the release pathway? Which pathways are used for viral egress? Why are certain cell lines not succeptible?