SEQUENTIAL PROPAGATION OF POLIOVIRUS RNA GENOMES USING IMPROVED TRANSFECTION

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Transfection procedures using poliovirus RNA have been improved to the point where the infectivity of the purified viral RNA approaches the infectivity of the intact virion. Using phenol extracted RNA with cationic detergent lipsomes and suspension cell cultures, we devised conditions that result in 100% infection of the cells. Previous transfection procedures were 10 to 100 less efficient. This high level of transfection, coupled with efficient RNA isolation procedures, provides a means for the sequential passage of poliovirus RNA while bypassing those viral functions involved in encapsidation and uncoating. Consequently, selective pressures result in the generation of subgenomic replicons retaining only those functions required for viral RNA replication. We are using this approach to follow the natural selective pressures on poliovirus defective interfering genomes when they are passaged in the absence of encapsidation constraints.