Nonhomologous RNA recombination in bovine viral diarrhea virus: Molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease

Four bovine viral diarrhea virus type 2 (BVDV-2) pairs consisting of cytopa thogenic (cp) and noncp BVDV-2 were isolated during an outbreak of mucosal disease. Comparative sequence analysis showed that the four noncp BVDV-2 is olates were almost identical. For the cp BVDV-2 isolates, viral subgenomic RNAs were shown by Northern blot to have a length of about 8 kb, which is a bout 4.3 kb shorter than the genome of noncp BVDV. Cytopathogenicity and th e expression of NS3 were both strictly correlated to the presence of viral subgenomic RNAs. By reverse transcription-PCR, Southern blot analysis, and nucleotide sequencing, a set of 11 unique subgenomes was identified with up to 5 different subgenomes isolated from one animal. To our knowledge, this is the first report on isolation of a set of pestiviral subgenomes from in dividual animals, common features of the BVDV-2 subgenomic RNAs include (i) deletion of most of the genomic region encoding the structural proteins, a s well as the nonstructural proteins p7 and NS2, and (ii) insertion of cell ular (poly)ubiquitin coding sequences. Three subgenomes also comprised 15 t o 75 nucleotides derived from the 5' part of the NS2 gene. comparisons of t he obtained nucleotide sequences revealed that the different-BVDV-2 subgeno mes evolved from the respective noncp BVDV-2: by RNA recombination. The pre sence of short regions of sequence similarity at several crossing-over site s suggests that base pairing between the nascent RNA strand and the accepto r RNA template facilitates template switching of the BVDV RNA-dependent RNA polymerase.