zika virus infection of fetal neural stem cells

Zika Virus Infection of Fetal Neural Stem Cells

Resuspend the pellet in ZIKV stock at a Multiplicity Of Infection, or MOI, of between 1 and 10. The volume of ZIKV solution should not exceed 0.5 milliliters. Incubate the samples at 37 degrees Celsius for one hour. After, invert the tube two to three times, and centrifuge the mixture at 216 times g at room temperature for five minutes to obtain a cell pellet.
Remove the supernatant by aspiration. Then resuspend the cells with DPBS to rinse them. Next, centrifuge the mixture at 216 times g for five minutes, and remove the supernatant by aspiration. Resuspend the cells with 12 milliliters of the appropriate medium. Then load 500 microliters of the infected cells into a 24-well plate.

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1. Zika Virus Infection
NOTE: ZIKV is highly sensitive to temperature, so it is imperative that ZIKV stock are stored at -80 &#176;C and freeze/thawing cycles are avoided. Keep working aliquots on ice.
<ol>
	<li>Passage cells. 3-4 million cells in total are needed to seed a 24-well plate, therefore when passaging, put the number of cells needed for infection and seeding in a flask when passaging.</li>
	<li>If seeding cells into a 24-well plate for staining, prepare the plate [Coat the wells containing cover slips with 0.01% poly-D-lysine (PDL) in sterile water and incubate for 1 h at 37 &#730;C. For a 24-well plate, use 250 &#956;L of PDL per well].</li>
	<li>After passaging, place 1 mL of cell suspension into a 1.5 mL microcentrifuge tube, and centrifuge at 200 x g at room temperature for 5 min. Then remove the supernatant by aspiration.</li>
	<li>Resuspend cell pellet in ZIKV stock to acquire a multiplicity of infection (MOI) of either 1 to 10. The volume of ZIKV solution should not exceed 0.5 mL. For mock treatment (control cells), resuspend cells in the same volume and type of medium used in ZIKV stock.</li>
	<li>Incubate above cell/ZIKV mixture at 37 &#176;C for 1 h. Invert the tube 2-3 times, and then centrifuge mixture for 5 min at 216 x g at room temperature to obtain a cell pellet. Resuspend pellet in dPBS and centrifuge again for 5 min at 216 x g at room temperature.</li>
	<li>Remove the supernatant by aspiration, resuspend cells with appropriate medium, and load infected cells into flask or plate needed for the experiment. If seeding the cells into a 24-well plate resuspend the pellet in 12 mL of appropriate medium, and then add 500 &#956;L of suspension to each well. 
	NOTE: At this point either maintain the cells in growth media to observe effects of ZIKV on stem cells, or go to steps 6 or 7 to study the effect of ZIKV on the differentiation process.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (10%)</td>
			<td>Sigma</td>
			<td>G8644</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Corning/cellgro</td>
			<td>25-060-c1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep (100x)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator</td>
			<td>Thermo Forma</td>
			<td>Model# 3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher Scientific</td>
			<td>75004221</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Forma scientific Claas II A/B3</td>
			<td>Model# 1284</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine hydrobromide(PDL)</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: McGrath, E. L., et al., <a href="https://app.jove.com/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
...

Take a suspension of the Zika virus, a pathogenic virus, having the desired multiplicity of infection, the ratio of virus particles to cells.
Mix the virus suspension with human fetal brain neural stem cells.
Incubate the mixture, allowing the virus to bind to receptors on the cells, facilitating viral endocytosis.
Next, centrifuge the mixture and discard the supernatant containing non-internalized viral particles.
Resuspend the virus-infected cells in media and seed them into an extracellular matrix protein-coated multi-well plate, allowing cell adhesion.
Incubate the plate.
The internalized Zika virus utilizes the host cell machinery to produce new viral particles.
Viral replication significantly alters cell morphology, causing cellular damage and facilitating the spread of infection to neighboring cells.
The Zika virus-infected cells can be used for further analysis.

10 ビュー. Zika Virus Infection of Fetal Neural Stem Cells

ビデオ: Zika Virus Infection of Fetal Neural Stem Cells - 実験

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.

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.

ジカウイルス感染細胞培養系と<em&gt;インビトロ</emインターフェロンの&gt;予防効果

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

JoVE Journal

免疫・感染学

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカ ウイルス感染症をモデル化

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

神経科学

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

脳内注入戦略を使用してジカ ウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Zika Virus Infection of Fetal Neural Stem Cells

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Zika Virus Infection of Fetal Neural Stem Cells