Here we report on a segment in the genomic 39 non-translated region (39NTR) of bovine viral diarrhea virus (BVDV) that is accessible for the insertion of foreign sequence elements such as the 59NTR of hepatitis C virus. Recombinant viruses exhibited replication kinetics similar to those of the parental strain, and characterization of RNA species after several passages revealed that foreign inserts had the same genetic stability as the BVDV 39NTR. The generation of such BVDV recombinants is relevant for several applications.Infections with bovine viral diarrhea virus (BVDV) elicit various diseases in cattle. For example, vertical infections may lead to persistently infected (PI) animals, which may die of mucosal disease (MD) when cytopathic (cp) BVDV biotypes emerge by mutation during virus replication .Within the family Flaviviridae, BVDV belongs to the genus Pestivirus, together with Classical swine fever virus (CSFV). The approximately 12 kb positive-strand RNA genome consists of a long open reading frame (ORF) and 59 and 39 non-translated regions (NTRs). Translation initiates at an internal ribosomal entry site (IRES) involving the 59NTR and the 59 portion of the ORF. The resulting polyprotein is processed by cellular and viral proteases to yield the viral structural and non-structural (NS) proteins (Lindenbach et al., 2007). In concert with cellular factors, the proteins NS3, NS4A, NS4B, NS5A and NS5B form viral replication complexes (RC) that catalyse the synthesis of progeny positive-strand viral RNAs via negative-strand RNA intermediates (Behrens & Isken, 2006).Studies on the molecular mechanisms of BVDV RNA replication were facilitated significantly by functional DNA copies of the viral genome. One of the first cDNAs that enabled in vitro transcription of infectious viral RNA was generated with BVDV CP7 (Meyers et al., 1996), a cp virus isolate from a PI animal that died of MD (Tautz et al., 1994). Considering its key role during viral RNA replication, the viral 39NTR has been particularly wellstudied. As with all pestiviruses, the BVDV 39NTR consists of a variable region (39V) downstream of the translational stop codon, and a conserved region (39C) at the 39 end (Deng & Brock, 1993) (Fig. 1). 39C contains inalterable elements that are assumed to compose the negative-strand promoter of the initial RC (Yu et al., 1999). Conversely, 39V displays a remarkable heterogeneity in size and nucleotide composition between different virus subtypes, and certain mutations within 39V are well-tolerated (Isken et al., 2004;Pankraz et al., 2005). Still, 39V also encodes conserved features, such as the so-called 'pseudo-stop codons', i.e. nucleotide triplets within the 39NTR that resemble translational stop codons and that are present in translational frame with the viral ORF. The presence of these 'pseudo-stops', as well as proper folding of the SL stop stem-loop structure in 39V and association of the cellular NFAR proteins with SL stop , were shown to be important for accurate termination of translation at the stop co...