Mixed triple: allied viruses in unique recent isolates of highly virulent type 2 bovine viral diarrhea virus detected by deep sequencing

Abstract

In February 2013, very severe acute clinical symptoms were observed in calves, heifers, and dairy cattle in several farms in North Rhine Westphalia and Lower Saxony, Germany. Deep sequencing revealed the coexistence of three distinct genome variants within recent highly virulent bovine viral diarrhea virus type 2 (BVDV-2) isolates. While the major portion (ca. 95%) of the population harbored a duplication of a 222-nucleotide (nt) segment within the p7-NS2-encoding region, the minority reflected the standard structure of a BVDV-2 genome. Additionally, unusual mutations were found in both variants, within the highly conserved p7 protein and close to the p7-NS2 cleavage site. Using a reverse genetic system with a BVDV-2a strain harboring a similar duplication, it could be demonstrated that during replication, genomes without duplication are generated de novo from genomes with duplication. The major variant with duplication is compulsorily escorted by the minor variant without duplication. RNA secondary structure prediction allowed the analysis of the unique but stable mixture of three BVDV variants and also provided the explanation for their generation. Finally, our results suggest that the variant with duplication plays the major role in the highly virulent phenotype.

Importance: This study emphasizes the importance of full-genome deep sequencing in combination with manual in-depth data analysis for the investigation of viruses in basic research and diagnostics. Here we investigated recent highly virulent bovine viral diarrhea virus isolates from a 2013 series of outbreaks. We discovered a unique special feature of the viral genome, an unstable duplication of 222 nucleotides which is eventually deleted by viral polymerase activity, leading to an unexpectedly mixed population of viral genomes for all investigated isolates. Our study is of high importance to the field because we demonstrate that these insertion/deletion events allow another level of genome plasticity of plus-strand RNA viruses, in addition to the well-known polymerase-induced single nucleotide variations which are generally considered the main basis for viral adaptation and evolution.

FIG 1

Survival of 8-week-old calves infected with the German BVDV-2c isolate NRW 19-13-8, with (n = 4) or without (n = 4) emergency vaccination at 5 days postinfection.

FIG 2

Schematic representation of p7- and NS2-encoding regions of the genomes of a prototypic BVDV-2c strain (Potsdam 1600), dup⁻ and dup⁺ variants from the recent BVDV infections in NRW, Germany, and the previously described highly virulent BVDV-2a strain 890. Corresponding parts of the genomes and the deduced proteins are depicted in corresponding colors (light and dark). (A) Structural variations at the genomic level, with the observed nonsynonymous mutations located within the duplicated region highlighted. (B) Implications of the genomic variations at the amino acid level, with the amino acid substitutions located within the duplication highlighted. The predicted transmembrane helices (TM) are shown as red arrows. Predicted cleavage sites are indicated by vertical arrows sized relative to the prototype cleavage site according to scores obtained with Emboss sigcleave.

FIG 3

Agilent Bioanalyzer 2100 analyses (with a DNA 7500 chip) of PCR products. (Upper panels) Gel view of Agilent traces. Bands representing dup⁺ and dup⁻ genomes are marked by arrows labeled accordingly. (Lower panels) Bar plots of relative quantities of PCR products determined by Agilent software, representing dup⁺ and dup⁻ genomes. (A) Analysis of recent BVDV-2c strains. NRW 19-13-8a (lane 7) depicts results for the NRW 19-13-8 isolate, reisolated from the animal trial. (B) Analysis of BVDV-2a 890 wild-type strain (v890WT) and clones. Results obtained for plasmids of the variants with (p890FL dup⁺) and without (p890FL dup⁻) duplication and the p890FL dup⁺-derived in vitro transcript are shown. In addition, results for virus rescued from in vitro-transcribed RNA and passaged in cell culture 2 (v890FL dup⁺ P2), 3 (v890FL dup⁺ P3), and 4 (v890FL dup⁺ P4) times are displayed.

FIG 4

Schema of theoretically possible dup⁻ variants and potential RNA secondary structures facilitating their generation. (A) Schematic representation of theoretically possible dup⁻ variants fitting the BVDV-2c prototype Potsdam 1600 that could be generated from dup⁺ genomes by deletions. Different shades of blue indicate different copies of the p7-NS2 region. (B) Circular graphs of RNA secondary structure predictions of the p7-NS2 region for the positive and negative strands. The outer circle represents the 5′-to-3′ sequence (clockwise). Arcs within the circle show base interactions (red, G-C; blue, A-U; and green, G-U). Along the circle, the modules of the duplicated region are highlighted. The sections marked in red represent interacting RNA stretches which bring corresponding regions of the two copies into close proximity, thereby providing an opportunity for the polymerase to circumvent exactly one copy. (C) Schematic straight representations of the circular parts of panel B, without base interactions.

FIG 5

Phylogenetic analysis (neighbor-joining tree with Tamura-Nei parameter [43]; gamma distribution = 3.71; 1,000 bootstrap replicates) of the open reading frames of different BVDV strains. Recent highly virulent isolates form a distinct subcluster within genotype 2c (highlighted in bold). Strain BVDV-2a 890 clusters within genotype 2a (highlighted in bold italics). Bootstrap values of >50 are indicated at the branches.