A Focus-Forming Assay for Quantifying Virus Titers in the Organs of an Infected Mouse

Take a multiwell plate containing a monolayer of mammalian cells.

Inoculate the wells with serial dilutions of tissue homogenates containing the Zika virus, derived from different organs of an infected mouse. 

Upon entry into the cells via endocytosis, the virus multiplies and buds off to infect neighboring cells.

Overlay with a high-viscosity methylcellulose solution, creating a physical barrier that restricts virus movement.

The barrier only allows infection of immediate neighboring cells, forming foci of infected cells, and ensures each focus originates from a single virus particle, enabling accurate quantification.

Fix the cells and permeabilize the membrane. Add primary antibodies that enter the cells and interact with the viral antigen.

Introduce a peroxidase-conjugated secondary antibody that binds to the primary antibody.

Add a chromogenic substrate that gets converted by peroxidase to create colored spots, indicating the foci of infected cells.

Count the foci to quantify the virus titer in the different organs of the mouse.