La Crosse (LAC) virus is an important cause of pediatric arboviral encephalitis in the United States. LAC virus is biologically transmitted by the mosquito Aedes triseriatus, and, like other arthropod-borne viruses, it establishes a persistent, nonpathogenic infection in its vector following oral infection. To investigate LAC virus persistent infection of mosquitoes, a reverse transcription-PCR assay was developed for the amplification of LAC virus negative-sense small (S) genome RNA segment, its full-length complement, and its mRNA transcript for qualitative analysis of transcription and replication in persistently infected mosquito tissues. RNAs were assayed from midguts removed at predetermined times after infection with a LAC virus-containing blood meal. LAC virus genome was detected almost uniformly in midguts at days 3 to 28 postinfection (p.i.) and, as the time p.i. progressed, in more of the samples than either mRNA or viral cRNA (vcRNA). Thus, persistent LAC virus infection of A. triseriatus midguts was correlated with a reduction in detectable viral mRNA and vcRNA. The assay was also used for analysis of virus-specified RNA in both quiescent and biosynthetically active mosquito ovaries. Viral replication decreased, as indicated by the absence of viral mRNA and vcRNA, in the ovaries of mosquitoes that did not receive further blood meals after their original oral infection. Viral replication increased in ovaries of mosquitoes that took an additional blood meal 30 days p.i. and was continuous in mosquitoes that took multiple meals to stimulate oogenesis. Thus, virus replication in persistently infected mosquito ovaries was dependent on host cell biosynthetic status.