This study investigates the infection of human fetal brain neural stem cells by the Zika virus. The methodology includes mixing the virus with the cells, allowing for viral endocytosis, and analyzing the effects of viral replication on cell morphology.
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.
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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