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. IMPORTANCEThis 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.

The Molecular Biology of Pestiviruses

“…However, more detailed analyses revealed that infection of cattle with BVDV-2 can be inapparent or accompanied by only mild symptoms and, vice versa, BVDV-1 is able to induce hemorrhagic disease (Lunardi, Headley, Lisboa, Amude, & Alfieri, 2008;Yesilbag et al, 2014). A very special case of fatal disease resulting from infection with a mixture of BVDV-2 was described recently (see Section 5) ( Jenckel et al, 2014).…”

“…Using a reverse genetics system of a BVDV-2a strain with this duplication, spontaneous deletions of this sequence were observed. A model described by the authors proposes an equilibrium between the two variants in infected animals ( Jenckel et al, 2014). It remains to be determined whether the viral variant without the duplication retains the highly virulent phenotype.…”

“…The figure was taken from Liu, Xia, Wahlberg, Belak, and Baule (2009). review. For more information on genotypes, we refer to corresponding publications ( Jenckel et al, 2014;Luzzago et al, 2014;Pan et al, 2005;Paton et al, 2000;Postel et al, 2012;Rosamilia et al, 2014).…”

Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus

“…▲ : Isolates from different clusters, but given the same name. References (I): [21,49,114,115,116]; (II): [41,49]; (III): [117]; (IV): [118]; (V): [119,120]; (VI): [52]; (VII): [21,49,121]; (VIII): [122,123,124,125,126]; (IX): [21]; (X): [127,128,129]; (XI): [21,49,130,131,132,133,134,135,136,137,138,139,140]; (XII): [141]; (XIII): [142,143]; (XIV): [144]; (XV): [21,49,52]; (XVI): [145,146]; (XVII): [21,40,147,148,149,150,151,152]; (XVIII): [153]; (XIX): [9,154,155,156]; (XX): [157,158,159,160,161,162]; (XXI): […”

Evidence for Tunisian-Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

“…Moreover, ruminant pestiviruses are genetically classified into various subtypes. To date, the viruses included in the BVDV‐1 species are differentiated into at least 20 subtypes, indicated as 1a‐1t (Giammarioli et al., ), BVDV‐2 consists of three subtypes named 2a–2c (Jenckel et al., ), and BDV includes at least eight subtypes designated BDV‐1 to BDV‐8 (Valdazo‐González et al., ; Dubois et al., ; Giammarioli et al., ; Peletto et al., ). Other pestiviruses have been isolated from batches of contaminated sheep pox virus vaccine in Tunisia and from sheep and goats in Turkey and Tunisia and genetically characterized (Thabti et al., ; Oguzoglu et al., ).…”