RNA recombination between persisting pestivirus and a vaccine strain: Generation of cytopathogenic virus and induction of lethal disease

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BV DV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a non cytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a lar ge internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are > 99% identical to the p reviously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orli ch, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses inc luding molecular cloning and nucleotide sequencing of the recombination par tners revealed that both homologous and nonhomologous RNA recombination con tributed to the generation of the viral subgenomes. Interestingly, for anot her cp BVDV isolate (CP 4584) from an independent case of MD, again an inse rtion of a RIT-derived sequence element was detected. In contrast to CP 174 1, for CP 4584 a duplication of the genomic region encoding NS3 and parts o f NS4A and NS4B was found. Transfection of bovine cells with RNA transcribe d from a chimeric cDNA construct showed that the RIT-derived insertion toge ther with the CP 4584-specific duplication of viral sequences represents th e genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of th e recovered cp virus in cell culture led to emergence of noncp BVDV and a n umber of viral subgenomes whose genome organization was similar to that in BVDV 1741.