Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493, Greifswald - Insel Riems, Germany.
BMC Vet Res. 2021 Jul 12;17(1):242. doi: 10.1186/s12917-021-02945-3.
As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5' untranslated region and the N protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation.
To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19-32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage.
Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.
牛病毒性腹泻病毒（BVDV）作为一种全球性反刍动物病原体，可引起牛病毒性腹泻病，其临床表现多样，在全球范围内造成严重的经济损失。该病毒属于瘟病毒属，其中 A 型和 B 型（同义 BVDV-1、BVDV-2）在基因上分化为 21 种 BVDV-1 和 4 种 BVDV-2 亚型。通常，5'非翻译区和 N 蛋白被用于亚型分析。然而，BVDV 的遗传变异性导致在分析基因组片段时，与全基因组评估相比，以前的研究存在局限性。
为了能够快速且易于进行 BVDV-1 和 BVDV-2 两种毒株的全基因组测序，我们对 12 个具有代表性的 BVDV 样本进行了纳米孔测序，这些样本是通过平铺 PCR 程序扩增得到的。这些样本涵盖了多种亚型（1b、1d、1f、2a、2c）、样本基质（血浆、EDTA 血液和耳缺口）、病毒载量（Ct 值 19-32）和物种（牛和羊），其中 10 个样本产生了全基因组序列，2 个低滴度样本的基因组覆盖率为 96%。
对新序列的进一步系统发育分析强调了全基因组测序对于识别新毒株和补充公共数据库中缺失序列信息的必要性。所提出的基于扩增子的测序方案允许快速、经济且易于获得完整的 BVDV 基因组。
