Flaviviruses are emerging threats to public health worldwide. Recently, one flavivirus, West Nile virus (WNV), has caused the largest epidemic of viral encephalitis in US history. Like other flaviviruses, WNV is thought to cause a persistent infection in insect cells, but an acute cytopathic infection of mammalian cells. To study adaptation of WNV to persistently replicate in cell culture and generate a system capable of detecting antiviral compounds in the absence of live virus, we generated subgenomic replicons of WNV and adapted these to persistently replicate in mammalian cells. Here we report that adaptation of these replicons to cell culture results in a reduction of genome copy number, and demonstrate that hamster, monkey, and human cells that stably carry the replicons can be used as surrogates to detect the activity of anti-WNV compounds. Additionally, we have used these cells to investigate the interaction of WNV genomes with interferon (IFN). These studies demonstrated that IFN can cure cells of replicons and that replicon-bearing cells display lower responses to IFN than their IFN-cured derivatives.