Zika Virus Infection of Fetal Neural Stem Cells

Abstract

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. 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.

Protocol

1. Zika Virus Infection

NOTE: ZIKV is highly sensitive to temperature, so it is imperative that ZIKV stock are stored at -80 °C and freeze/thawing cycles are avoided. Keep working aliquots on ice.

1. 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.
2. 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 ˚C. For a 24-well plate, use 250 μL of PDL per well].
3. 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.
4. 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.
5. Incubate above cell/ZIKV mixture at 37 °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.
6. 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 μL 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Gibco	11965-092
F12	Gibco	11765-054
Glucose (10%)	Sigma	G8644
HEPES (1M)	Corning/cellgro	25-060-c1
Pen Strep (100x)	Gibco	15140-122
Carbon dioxide incubator	Thermo Forma	Model# 3110
Centrifuge	Thermo fisher Scientific	75004221
Biological safety cabinet	Forma scientific Claas II A/B3	Model# 1284
Poly-D-Lysine hydrobromide(PDL)	Sigma	P6407-5MG
Laminin	Invitrogen	23017-015