A universal 'One-tube' RT-PCR protocol for amplifying isolates of bovine viral diarrhoea virus

The increase in the knowledge of the genetic variability of BVDV and the id entification of some of the genetic determinants of its pathogenicity requi re robust and practical tools for rapid molecular characterization of the v arious genotypes of this virus. This study was undertaken to develop a stan dard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction . The reaction set-up is very flexible because it consists of two pre-mixes . These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any pr imer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amoun ts sufficient for subsequent sequencing reactions. The method was applied t o five different regions of the BVDV genome: (i) the well-known 5'-UTR to d ifferentiate genotypes I and II; (ii) the entire E2 gene, or an approximate ly 550 bp region within the E2 gene, in order to find the molecular equival ent of antigenic varieties; (iii) the entire structural protein coding regi on covering the Npro, capsid, E-RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic bi otypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B gene s. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from ei ther 10(6) infected bovine embryonal lung cells or the same number of leuko cytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis v irus/BVDV recombinants. The correct processing of the amplicon-derived E2 g lycoprotein of BVDV strain PT810 was demonstrated by its reaction with a mo noclonal antibody in an immunofluorescence assay. Given the variety of RT-P CRs tested, we conclude that this universal protocol may be useful with oth er RNA viruses.