Isolation of a replication-efficient mutant of West Nile virus from a persistently infected genetically resistant mouse cell culture.

Flavivirus-resistant mouse embryofibroblasts (C3H/RV) that were infected with West Nile virus, strain E101 (WNV), yielded fewer infectious virions than did cultures of congenic susceptible cells (C3H/HE). Analysis of intracellular viral RNA synthesis indicated that the incorporation of [3H]uridine into 40S genome RNA was markedly reduced in resistant cells, and about 100-fold less labeled 40S RNA was found in pelleted extracellular virions from resistant cultures than in those from susceptible ones. A non-temperature-sensitive mutant of WNV isolated from culture fluid of a persistently infected culture of genetically resistant mouse cells was found to produce higher yields of infectious virus than the parental WNV used to initiate the persistent infection. Analysis of intracellular actinomycin D-resistant RNA indicated that the mutant virus (WNV-RV) was more efficient at incorporating [3H]uridine into 40S RNA in resistant cells than was the parental virus. WNV-RV also synthesized 40S RNA more efficiently than parental virus in congenic susceptible cells and in BHK cells. Analysis of the incorporation of [35S]methionine into viral proteins was likewise enhanced in WNV-RV-infected cells. The WNV-RV mutant provides a tool for studying the regulation of transcription of flavivirus RNA.