Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells

An assay for the bovine viral diarrhoea virus (BVDV) replicase was develope d using extracts from BVDV-infected cells. The replicase activity was maxim al approximately 8h post-infection as measured by the generation of a genom ic length radiolabelled RNA. Using a semi-denaturing gel system, three viru s-specific in vitro radiolabelled nascent RNA species were identified. A fa st-migrating RNA was demonstrated to be the double-stranded replicative for m (RF). A second form was shown to be a partially single-stranded/partially double-stranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was depe ndent on 5-10 mM Mg2+ and although it was also active in 1-2 mM Mn2+ it was inhibited at higher concentrations. The optimum KCI concentration for labe lling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative m odel of BVDV RNA replication.