Abstract
In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.