Abstract
BackgroundBovine 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.MethodsIn 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.ResultsWe 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.ConclusionsBy 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.
Highlights
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 primer set used for the amplification of complete BPV genomes in batch 1 (V1) consisted of ten overlapping amplicons, divided across two pools, that spanned the complete BPV genome
Comparable DNA amplification was observed in all twelve samples, but only for eleven samples, at least one complete genome sequence could be recovered from the nanopore data
Summary
Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. Sarcoids are non-metastasizing, but persistent tumours of fibroblastic origin with a wide range of clinical entities, often occurring simultaneously within the same individual [3] According to their gross morphology, Gysens et al Virology Journal (2022) 19:8 five sarcoid types are described: occult, nodular, verrucous, fibroblastic and mixed, with the latter being a combination of several of these types [4, 5]. Members of the family Papillomaviridae typically share a similar genome organization, characterized by a double-stranded DNA genome of approximately 8 kbp containing a non-coding long control region (LCR) and eight open reading frames (ORFs) These ORFs encode six early proteins (E1–E2, E4–E7) and two late proteins (L1, L2). Some RNA molecules containing late transcripts have been demonstrated within sarcoids [12, 13], yet it remains unclear whether this culminates in the production of infectious virions
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