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

Abstract

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large 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 previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, 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 including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.

FIG. 1

Analysis of BVDV 1741. (A) Northern blot analysis of total RNA from MDBK cells infected with CP 1741, NCP 1741, and noninfected MDBK cells (n.i.). RNA was separated by denaturing agarose gel electrophoresis, blotted onto a nylon membrane and hybridized with a 2.5-kb NotI-NsiI fragment from the cDNA clone pCP7-5A (7). Numbers refer to RNA ladder sizes. Migration positions of the viral genomic and subgenomic RNAs are marked with arrows. (B) Genome organization of NCP 1741 and the CP 1741 subgenomic RNAs. All subgenomic RNAs contain an insertion of CP RIT-derived sequences which encode part of S27a (S27a∗) fused to an N-terminally truncated ubiquitin (ubi∗) (black box) as well as flanking viral sequences (gray boxes). The deletions are indicated by dashed lines. The lengths of the bars are not drawn to scale. The underlined parts of the (sub)genomes have been sequenced. With respect to the analyzed regions, all subgenomes maintain one large ORF.

FIG. 2

Analysis of cp and noncp BVDV 4584. (A) Northern blot analysis of total RNA from MDBK cells infected with BVDV CP 4584, NCP 4584, and noninfected MDBK cells (n.i.). RNA ladder sizes are indicated on the left. Migration positions of the viral genomic RNAs are marked with arrows. (B) Genome organization of NCP 4584 and CP 4584. The genome of CP 4584 contains a duplication of viral sequences encoding NS3, NS4A, and part of NS4B together with an insertion of CP RIT-derived sequences which encode S27a∗ and ubi∗ (black box) as well as flanking viral sequences (NS4B∗, NS3∗; gray boxes). The lengths of the bars are not drawn to scale. The underlined parts of the genomes have been sequenced.

FIG. 3

Schematic representation of part of the genome of BVDV vaccine strain RIT (top) and the RIT-derived sequence elements identified in the subgenomes A to D of CP 1741 and in the genome of CP 4584. The RIT-derived sequence elements encode S27a∗ and ubi∗ (black box) as well as flanking viral sequences (NS4B∗, NS3∗; gray boxes). The lengths of the bars are not drawn to scale. The numbers of amino acids corresponding to the indicated proteins or parts thereof are shown below the bars.

FIG. 4

Transfection experiments with RNA transcribed from infectious cDNA clones pNCP7-5A, pCP7-5A, and p7/4584. (A) Genome organization of noncp BVDV strain NCP7-5A and the chimeric cDNA construct p7/4584 that mirrors the genome structure of CP 4584. Generation of p7/4584 was based on cDNA clone pNCP7-5A-(AgeI-), which differs from the infectious noncp cDNA clone pNCP7-5A by the absence of a single AgeI site. For 7/4584, the CP 4584-specific part of the genome encompassing part of the CP RIT-derived insertion (gray and black boxes) and positions of the MluI (■) and AgeI (▴) sites used for cDNA cloning are indicated below the bars. The remaining part of p7/4584 was derived from pNCP7-5A-(AgeI-). The lengths of the bars are not drawn to scale. (B) Northern blot analysis of total RNA from MDBK cells infected with the indicated viruses at an MOI of 0.05. The infected cells were processed in parallel with those used to determine the growth rates (C). RNAs were extracted at 24 h after infection. Northern blotting was performed as described in the legend for Fig. 1A. Numbers refer to RNA ladder sizes. Migration positions of the viral genomic RNAs are marked with arrows. The intensity of bands was determined with a phosphorimager. The relative amounts of viral genomic RNAs are indicated below the blot (percentage of CP7-5A value [100%]) n.i., RNA from noninfected MDBK cells. (C) Growth curves of the recovered BVDV CP7-5A, NCP7-5A, and 7/4584 determined on MDBK cells infected at an MOI of 0.05. The titers of released virus were determined over a 5-day period. (D) Plaques produced by BVDV CP7-5A and 7/4584 and foci produced by BVDV NCP7-5A at 4 days after infection of MDBK cells. For infection, dilutions of the supernatants from cells transfected with CP7-5A RNA, NCP7-5A RNA, and 7/4584 RNA were used. Infection of cells was visualized by immunostaining.

FIG. 5

Emergence of noncp BVDV and subgenomic RNAs after passages of cp BVDV 7/4584 in MDBK cells. (A) Northern blot of total RNA from MDBK cells infected with the indicated passage number (passage 1 [P1] and P5 to P10) of 7/4584 at an MOI of about 0.1. RNAs were extracted at 48 h after infection. Northern blotting was performed as described in the legend for Fig. 1A. Numbers refer to RNA ladder sizes. Migration positions of the genomic 7/4584 RNA (upper arrow), the genomic RNA of the emerged noncp BVDV (middle arrow), and the emerged subgenomic RNAs (lower arrow) are indicated on the right. (B) Genome organizations of BVDV 7/4584 and the subgenomic RNAs 7/4584-A (B to F) which evolved after 8 and 10 passages of 7/4584 in MDBK cells. The CP RIT-derived sequences are marked by gray and black boxes. Deletions are indicated by dashed lines. The lengths of the bars are not drawn to scale. The sequenced regions of the subgenomes are indicated by a line below the bar at the bottom. With respect to the analyzed regions, all subgenomes maintain one large ORF. The genome organization of the emerged noncp BVDV is not included.