Quantification of Infectious Hepatitis C Virus by Fluorescence Microscopy

Begin with a mammalian cell culture supernatant containing Hepatitis C virus.

Serially dilute the viral suspension in growth medium.

Inoculate each dilution onto a monolayer of hepatoma cells and incubate.

The virus enters the cells, releases its genome, and expresses viral proteins, including the non-structural replication protein.

This produces new viral particles that bud off and infect neighboring cells, forming a foci of infected cells.

After incubation, remove the medium and add a fixative. Incubate at cold temperature to fix the cells and permeabilize the membrane.

Wash and add a blocking buffer to prevent non-specific antibody binding.

Introduce primary antibodies that bind to the non-structural replication protein.

Wash to remove unbound antibodies.

Then, introduce fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Wash again to remove unbound antibodies. Add a DNA-binding dye to stain the nuclei.

Visualize under a fluorescence microscope and quantify the fluorescent foci to determine the virus concentration of the supernatant.

Plate naive Huh-7.5.1 cells at approximately three times 10 to the third cells per well using a 96 well plate. The next day, perf