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 developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (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 dependent 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 labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.