An Assay for Measuring the Relative Replication Fitness of Viral Strains

Take a culture of human blood immune cells. Add a mixture of equal volumes of a prototype viral strain and a mutant strain.
Reserve a portion of the inoculum to measure the initial strain ratio.
During incubation, the virus enters the host cells and releases its genome into the cytoplasm.
Next, replace most of the medium. Then, centrifuge and discard the supernatant to remove any unbound virus.
Add fresh medium and incubate. In the infected cells, the viral genome is expressed to produce new virions, which are released into the medium.
At regular intervals, collect a portion of the medium, centrifuge it, and store the supernatant containing the virions for quantification.
Replenish the culture with a fresh medium and incubate it to continue viral production.
Plot the viral strain ratios over time. Compare the initial and subsequent viral strain ratios. A progressive increase in the proportion of one viral strain indicates higher relative replication fitness than the other strain.

To prepare for the growth competition assay, seed three times 10 to the fifth PHA stimulated peripheral blood mononuclear cells or PBMCs in a total volume of 500 microliters per well in a 48-well flat-bottom plate