Name: zika virus infectious cell culture system and the in vitro prophylactic effect of interferons
Uploaded: 2016-08-23T15:00:00
Description: Watch this Scientific Journal Video about Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons at JoVE.com

We would like to thank Dr. Aaron Brault and Dr. Brandy Russell of the Centers for Disease Control and Prevention (CDC), USA for providing Zika viral strain PRVABC59. We thank Nicholas Ten of Yale University for copy-editing this manuscript. This work was supported by the Cedars-Sinai Medical Center Institutional Programmatic Research Award to V.A.

Note: A schematic outline of the work flow is presented in Figure 1.
1. Cells
<ol>
	<li>Use Vero cells for Zika virus production and analysis of viral replication cycle.</li>
	<li>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.</li>
	<li>Culture Vero cells with the specified complete growth medium at 37 &#176;C with 5% CO2.</li>
</ol>
<p class="jove_ti...

<ol>
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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...

Zika Virus (ZIKV) is an important human pathogen associated with microcephaly and poor pregnancy outcomes1,4. 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 reported5,6. 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 Zika forest, Uganda and the first human case was reported in 19527,8. Individuals that become infected with ZIKV present with mild symptoms such as fever, rash, headache, conjunctivitis, and muscle/joint pain. Infected pregnant women can transmit ZIKV to the developing fetus1. ZIKV infection has also been linked to Guillain-Barre syndrome, a peripheral nerve auto-immune demyelination disorder9.
The Zika viral genome consists of a positive sense, single-stranded RNA molecule which is about 10.8 kilobases in length. The genome&#39;s structure is organized as 5&#39;NCR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-3&#39;NCR, with non-coding regions (NCR) flanking a protein-coding region6. A single polyprotein (3,419 aa) is translated that is co- and post-translationally cleaved into 10 smaller peptides. Both the 5&#39;NCR and 3&#39;NCR RNA stem-loop structures play a critical part in the commencement of viral genome translation and replication. The structural components of the genome are comprised of the capsid, membrane, and envelope proteins. The non-structural proteins are critical for genome replication.
Currently, Zika viral strains are grouped into three main genotypes: West African, East African, and Asian6,10-13. It has been proposed that the East African lineage spread to West Africa and Asia, where it later further evolved12. The Asian genotype is responsible for the current outbreaks in the Americas. Zika virus can be cultured in both mosquito and mammalian cells. Primary dermal fibroblasts, immature dendritic cells, cortical neural progenitor cells, and Vero cells are susceptible to Zika viral infection10,14,15. Both type I and type II interferons have been shown to restrict ZIKV growth in skin fibroblasts15. The objectives of this study are to provide a step-wise, detailed protocol for the production and assaying of the Asian genotype ZIKA viral strain PRVABC59 in a mammalian cell culture system and to demonstrate the utility of this infectious culture system as a drug development platform. This resource has the potential to greatly benefit the Zika viral and neurological research community to further elucidate its mechanisms of viral pathogenesis and evolution of viral virulence.

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			<td height="21" style="height:21px;width:569px;">Countess-cell counting&#160; chamber slides</td>
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			<td height="21" style="height:21px;width:569px;">Vero Cell Line&#160;</td>
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			<td height="22" style="height:23px;width:569px;">Zika viral strain PRVABC59</td>
			<td style="width:355px;">Centers for Disease Control and Prevention (CDC)</td>
			<td style="width:119px;"></td>
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			<td height="22" style="height:23px;width:569px;">Interferon alpha A&#160;&#160;</td>
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zika virus infectious cell culture system and the in vitro prophylactic effect of interferons

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-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; 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.

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...

Watch this Scientific Journal Video about Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons at JoVE.com

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

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.

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

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

The overall goal of this study is to establish a mammalian cell-based in vitro Zika virus infectious culture system to study Zika virus replication, as well as utilization of the in vitro platform for drug development. This step-by-step detailed protocol of Zika virus infectious cell culture system can help address the mechanism of Zika virus mediated disease pathogenesis and evolution of viral virulence. The main advantage of this technique is that it can allow for large-scale production of Zika virus for drug and vaccine development.Begin by harvesting Vero cells at 80%cell density in a T75 culture flask by aspirating the medium from the flask and rinsing the cells with two milliliters of phosphate-buffered saline. Add two milliliters of 0.25%trypsin and incubate the flask at 37 degrees Celsius for five minutes. Following the incubation, add eight milliliters of growth medium containing serum to inactivate the trypsin.Then, transfer the cells to a 15 milliliter tube. Next, remove 10 microliters of cells and mix with an equal amount of 0.4%trypan blue. Load the prepared mix to the cell counting chamber slide and obtain account of the viable cells using an automated cell counter.Seed a total of seven-million cells into a T160 flask in a 30 milliliter volume. The next day, prepare an appropriate multiplicity of infection of Zika virus inoculum in 10 milliliters of serum-free culture medium per flask. Safe handling of the virus is critical to prevent the spread of infection.Remove the spent medium from the T160 flask and then add the full 10 milliliters of freshly prepared viral inoculum. Incubate the inoculated flasks at 37 degrees Celsius with 5%CO2 for four to six hours. Spread the inoculum by gently tilting the flask sideways every hour of the incubation.At the completion of the incubation, replace the inoculum with 30 milliliters of warm serum-supplemented growth medium. Then, continue the viral culture for the next 96 hours. After four days, verify the progression of infection by observing the appearance of viral plaques on the cell monolayer, using a phase contrast microscope.Take images as needed at 40x and 200x magnification. Harvest cell-culture supernatants at the 96 hour time point and centrifuge the supernatant at 300 times G for 10 minutes at four degrees Celsius to remove cell debris. After centrifugation, carefully remove the supernatants without disturbing the pelleted debris, and transfer them into 15 milliliters tubes.Store the viral culture supernatants in multiple aliquots at negative 80 degrees Celsius. To titer the virus, first seed naive Vero cells into a 12-well plate at one times 10 to the fifth cells per well, in a two milliliter volume. The following day, prepare tenfold serial dilutions of viral culture supernatants, collected from the T160 flask using serum-free medium.When preparing serial dilutions, a viral culture supernatant for titer acids, make sure to safely handle the virus to prevent infection. Next, remove the spent medium from each well of the 12-well plate containing Vero cells, and add 400 microliters of the diluted inoculum onto Vero cells in triplicate. Incubate the inoculated plates in the tissue-culture incubator for four to six hours.At the end of the incubation, replace the inoculum with two milliliters of serum-supplemented medium per well. At 48 hours post inoculation, count the viral foci at the appropriate dilutions, using a phase contrast microscope. Calculate the viral titer as plaque-forming units per milliliter.After inoculating Vero cells in a 12-well plate with high and low titers of Zika virus, and incubating for 48 and 96 hours, genome replication is assessed by quantitative PCR for expression of the Zika virus genome, and by immunocytochemistry for double-stranded RNA. For immunocytochemistry, remove the medium from the wells of the 12-well plate, and add one milliliter of methanol to each well. Allow the cells to fix at four degrees Celsius for 30 minutes.Next, wash the fixed cells three times with 1x PBS. Then add blocking buffer and incubate for one hour at room temperature. Dilute the mouse monoclonal anti-double-stranded RNA antibody J2 one to 100 in blocking buffer.Add it to the cells, and incubate the cells in this solution overnight at four degrees Celsius. The next day, dilute goat anti-mouse IgG-594 secondary antibody one to 1, 000 in blocking buffer, and after washing the cells with 1x PBS, add it to the cells, and incubate them for one hour at room temperature. After washing the cells with 1x PBS, stain for nuclei using hoechst dye, and observe the cells using a fluorescent microscope at 100x magnification.Six hours after seeding a 12-well plate with naive Vero cells, as before, add cytokines of interest at indicated concentrations in biological duplicates. 12 hours posttreatment, perform Zika virus infection, and include negative-control wells without infection. Four hours after infection, replace the viral inoculum with cytokine-treated medium and then incubate the cells at 37 degrees Celsius for an additional 44 hours.At 48 hours post infection, count viral plaques using a phase contrast microscope, and acquire representative images of infected cells at 40x magnification. This graph shows Zika virus genomic content measured by RT-qPCR, both Pan genotype and PRVABC-59 strain primers showed equal sensitivity and specificity. As expected, hepatitis-C viral primers did not amplify any product.Zika viral growth kinetics at the indicated time points in low and high titer infected cells, compared to that of the uninfected mock control, are shown here. Results from the immunocytochemistry assay show that at 48-hours post infection, the infected cells are in a few clusters, whereas at 96-hours post infection, most of the cells are infected. This graph shows modulation of Zika viral growth by various cytokines, compared to that of the vehicle control.These phase-contrast images show Zika virus-infected cells with or without cytokine treatment. Viral plaques formed by infected cells can be seen as foci. Note that interferon treated wells do not have viral plaques.Following this procedure, other methods such as Zika virus high-resolution domain mapping for viral protein localization and replication in mosquito and human cells can be performed to answer additional questions, such as elucidation of the mechanism of disease pathogenesis and evolution of viral virulence. After watching this video, you should have a good understanding of how to establish the mammalian cell-based in vitro Zika virus infectious culture system to study Zika virus replication, as well as the placation of in vitro platform for drug and vaccine development.

Research

JoVE Journal

Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...