The noncytopathic Australian bovine viral diarrhoea virus (BVDV) Trangie isolate was used to establish a one-step growth curve and to investigate previously uncharacterised aspects of pestivirus replication. The regions of the genome corresponding to the 5'UTR and the NS3 gene of BVDV were amplified by RT-PCR and then cloned and sequenced. The predicted amino acid sequence of NS3 shows about 97% homology with that of the NADL and SD-1 strains, whereas the nucleotide sequence of the 5'-UTR is 95.1% homologous with the NADL and 91.2% with SD-1 strains, respectively. A one-step growth curve established in BTu cells using a moi of 20 showed that the eclipse phase was approximately 8 to 10 h and the first appearance of viral antigen assayed by immunofluorescence occurred around 6 h post infection (p.i.). Both positive and negative sense virus RNAs were first detected at 4 h p.i. by Northern blot hybridisation using strand-specific RN A probes generated by in vitro transcription from the NS3 clone. The ratio of positive : negative sense virus RNA changed from 2 : 1 at 4 h p.i. to 10 : 1 at 12 h p.i. and thereafter. The kinetics of synthesis showed that the rate of synthesis of positive strand viral RNA increased rapidly from 6 h p.i, whereas the rate of synthesis of the negative strand remained constant. The copy number of genomic RNA determined by Northern blot hybridisation analysis was estimated to be l.5x104 copies per cell, 16 to 24 h p.i.. The replication of bovine viral diarrhoea virus (BVDV) RNA is considered to involve replicative intermediates (RI) and replicative forms (RF) of virus RNA. Viral RNA species in BVDV-infected cells that were thought to represent replicative intermediate (RI) and replicative forms (RF) were detected after electrophoretic separation by urea-PAGE. Confirmation of the identity of the RI and RF was obtained using lithium chloride precipitation and RNase A digestion of 3H-uridine labelled RNA. Pulse-chase labelling of BVDV-infected cells was consistent with synthesis of nascent BVDV RN A through a RI derived by strand displacement from a RF template. The structure of the BVDV RI was examined and calculated to contain 6 nascent strands on the template, based on the RNase-resistance after separation of the radiolabelled RNA species by sucrose gradient centrifugation and digestion of the fractions with RNase A in high salt (2xSSC). Thus the synthesis of BVDV RNA is likely to be similar to the model proposed for flavivirus replication. Bovine turbinate (BTu) and lamb testis (LT) cell lines persistently infected with bovine viral diarrhea virus (BVDV) arose as a result of a single change of medium containing commercial fetal calf serum. Infected cells comprised 30% and 50% respectively, of the total cell population, determined by immunohistochemical staining. The ratio of positive cells has remained unchanged during successive passages. Characterisation of the persistently­ infected BTu cells (BTuI) showed that only full length viral RNA was detected by Northern blot hybridisation, proving that DI particles were not involved. Secreted and intracellular virus from these cells were fully infectious for fresh BTu and LT cells. The BTuI cell line was fully permissive for a cytopathic BVDV isolate and a bovine herpesvirus, but non-permissive for two non-cytopathic BVDV isolates. Attempts to induce the permissive state in the BVDV-negative cells of the BTul culture by treatment with actinomycin D and 5'-aza-cytidine failed. These cells provide a convenient model to study aspects of BVDV pathogenesis and replication.