## Calculation and comparison of viral growth rates in competitions

To compare and evaluate competitive viral fitness, the viral growth rate (g, a combined growth/decay
parameter for viral production) of each virus in a competition as well as the growth rate difference
(d) and ratio (r) of the two viruses are calculated with 95% confidence intervals based on the
mathematical model below:

*V(0)*is the number of infectious particles in the inoculum at time zero, and *V(t)* is the free viral production
at time *t* after infection. *m* and *f* are two competing viruses, *m* referring to the mutant virus and *f*
referring to the founder or wild type virus.

In addition, if *m* and *f* viruses are in same viral background (e.g. NL4-3) but with different tags
(e.g synonymous nucleotide differences introduced into a certain gene of the viral background),
to account for any growth differences caused by the differences in the tags and assay variations
between different experiments, estimates of *d* and *r* from competitions between *m* and *f* (experimental)
can be compared to those estimated from competitions between *f* viruses with different tags (control).
If *p*<0.05, the values of *d* or *r* in the experimental and control conditions were considered to be
significantly different.

Finally, in order to analyze repeat replicates simultaneously, the estimation procedures utilize Generalized Estimating Equations
(GEE) to account for correlation within replicates.

Please cite the following paper if you use Viral Growth Rate Calculation:

Yi Liu, Sarah Holte, Ushnal Rao, Jan McClure, Philip Konopa, J. Victor Swain, Erinn Lanxon-Cookson, Moon Kim, Lennie Chen,
and James I. Mullins. A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the
competitive replication fitness of human immunodeficiency virus type 1 in cell culture.
Journal of Virological Methods. 2013, 189:157-66.