Japanese encephalitis virus replication: A procedure for the selective isolation and characterization of viral RNA species

The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 106 to 107 p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 μg AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.