Recent studies have shown that West Nile virus (WNV) can induce an asymptomatic persistent infection in the kidneys of experimentally infected hamsters. The chronically infected rodents shed virus in their urine for up to 8 months, despite the disappearance of viremia and the development of high levels of neutralizing antibodies. WNV, like most members of the Japanese encephalitis virus complex (Flavivirus; Flaviviridae), is assumed to be mainly neurotropic; little is known about the genetic basis for its renal tropism. In this study, complete sequence analyses were done to compare four WNV isolates from the urines of persistently infected hamsters with the wild-type parent virus (NY 385-99). Nucleotide changes, ranging from 0.05% to 0.09%, were identified in all of the WNV isolates from urine; most of the changes were in coding regions, causing amino acid substitutions in the E, NS1, NS2B, and NS5 proteins. The genetic changes associated with renal tropism were also accompanied by a loss of virulence for hamsters and a change in plaque morphology.