Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium.
Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven - University of Leuven, Herestraat 49/Box 1040, 3000, Leuven, Belgium.
Virol J. 2022 Jan 6;19(1):8. doi: 10.1186/s12985-021-01735-5.
Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation.
In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees.
We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis.
By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.
牛乳头瘤病毒（BPV）1 型和 2 型在马最常见的肿瘤——马肉瘤的病因学中起着核心作用。一方面，在临床表现上存在独特的多样性，而另一方面，BPV-1 感染的宿主临床表现也存在依赖性，这表明存在其他因素。早期的研究已经报道了同种型内序列变异的潜在功能意义，以及源自肉瘤的 BPV 变异的存在。因此，同种型内序列变异似乎是一个重要的新兴病毒因素。本研究旨在广泛了解源自肉瘤的 BPV 变异，并探讨其与疾病表现的潜在关联。
为了实现这一目标，我们成功优化了纳米孔测序方法，用于筛选广泛的临床样本。首先，通过定量实时 PCR 对每个肿瘤样本进行 BPV-1/-2 筛查。在 BPV 阳性样本中使用定制的引物组，通过两个多重 PCR 反应扩增完整的病毒基因组，产生一组重叠的扩增子。为了进行系统发育分析，对所有可用的 BPV-1/-2 完整基因组序列分别进行了单独的比对。使用生成的比对推断贝叶斯系统发育树。
我们发现源自肉瘤的 BPV-1 存在大量遗传变异，但这种变异与疾病严重程度无关。一些 BPV-1 基因组存在多个主要缺失。值得注意的是，它们中的大多数都聚集在编码晚期病毒基因的区域内。再加上所描述的缺失的广泛程度（多达 603 个核苷酸），这表明 L1/L2 在疾病发病机制中的功能发生了改变。
通过生成大量完整的 BPV 基因组，我们成功地将下一代测序技术引入兽医研究，重点关注马肉瘤，从而首次报道了基于纳米孔的完整源自肉瘤的 BPV-1/-2 测序以及来自单个临床样本的多个完整基因组的同时纳米孔测序。
