Characterization of West Nile virus persistent infections in genetically resistant and susceptible mouse cells I. Generation of defective nonplaquing virus particles

Abstract

Persistent infections with West Nile virus (WNV), strain E101, were readily established in SV40-transformed mouse embryofibroblast cell lines. Four separate persistently infected cultures maintained at 37° by weekly subculture, spontaneously stopped producing infectious virus as assessed by plaque assay on BHK cells by the sixteenth week. In contrast, duplicate cultures switched to 32° after the sixth subculture continued to produced virus for an extended period. However, cells that no longer produced detectable plaquing virus remained positive for WNV-specific immunofluorescence and were resistant to superinfection with WNV. Although attempts to rescue plaquing virus were unsuccessful, flavivirus-like particles were observed in these cells by electron microscopy within dense cytoplasmic vacuoles and in culture fluids. Analysis of RNA within the extracellular particles produced by these cells revealed the presence of several species of RNA all smaller than the 40 S genome RNA of wild type WNV. This RNA was demonstrated to be WNV-specific by hybridization with DNA complementary to wild type WNV 40 S RNA. The extracellular particles were able to infect BHK cells, but their ability to interfere with the replication of wild type WNV was found to be low. The frequency of generation of nonplaquing WNV mutants by the mouse cell lines indicates that these cells may exert a selective pressure for their enrichment.