Molecular Detection by Rolling Circle Amplification Combined with Deep Sequencing of Mixed Infection by Bovine Papillomaviruses 2 and 4 in Carcinoma In Situ of the Bovine Esophageal Mucosa

Abstract

Papillomaviruses (PVs) are oncogenic and infect the skin and mucosa of various host species. Considering the recent advances in research on PVs using rolling circle amplification (RCA) followed by high-throughput sequencing (HTS), in this study, we aimed to investigate the bovine papillomavirus (BPV) types associated with proliferative lesions in the upper alimentary tract of an affected bull and characterize the viral strains through complete genome sequencing using this strategy. We analyzed the PV strains associated with two hyperplastic esophageal lesions through PCR using degenerate primer pairs and RCA, followed by HTS. HTS of the libraries generated using RCA products provided the whole genome sequence of BPV4 present in squamous papilloma, whereas the complete genome sequence of BPV2 and subgenomic fragments of BPV4 were identified in carcinoma in situ (CIS). For the first time, we have sequenced BPV2 identified from the CIS of the bovine upper alimentary canal. Additionally, RCA followed by HTS allowed characterization of the mixed infection by BPV2 and BPV4 in this lesion. These data reveal that BPV4 is not the only BPV type present in CIS of the esophageal mucous membrane; moreover, a mixed infection caused by BPV2 and BPV4 at the tested anatomical site was demonstrated.

Keywords: cattle; coinfection; esophagus; histopathology; next generation sequencing; papilloma; polymerase chain reaction; upper alimentary tract.

Figure 1

Esophageal mucosa of a Girolando dairy bull. (A) Marked epithelium proliferation and parakeratotic hyperkeratosis resulting in exophytic folding. Scale bar 200 μm; (B) acanthosis and koilocytosis (arrows). Scale bar 20 μm; (C) proliferation of disorganized epithelial cells without invasion through the basement membrane into the submucosa. Scale bar 100 μm; (D) epithelial proliferation without invasion of the basement membrane (arrows). Scale bar 10 μm. Figure (A–D)—hematoxylin–eosin.

Figure 2

Phylogenetic analysis of the Brazilian bovine papillomavirus strain identified in a squamous papilloma developed in the esophagus of a dairy bull from southern Brazil using complete L1 gene nucleotide sequences. The evolutionary history was inferred using the maximum likelihood method and the general time reversible model. The tree with the highest log likelihood (−18,441.70) is shown. Numbers at internal nodes represent the bootstrap support values (percentages) determined for 1000 replications (values < 50 are not shown). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. We included 18 nucleotide sequences in this analysis. There were a total of 1580 positions in the final dataset. Evolutionary analyses were conducted using MEGA11 [26]. The Brazilian bovine papillomavirus strain sequenced in this study is indicated using a black-filled circle. Representatives of the viral PV types belonging to the five species of the genus Xipapillomavirus, recognized by the International Committee on Taxonomy of Viruses (ICTV), are shown. The GenBank accession numbers of the PV types included in the phylogenetic analysis are indicated.

Figure 3

Phylogenetic analysis of the Brazilian bovine papillomavirus strain identified in a carcinoma in situ lesion developed in the esophagus of a dairy bull from southern Brazil using complete L1 gene nucleotide sequences. The evolutionary history was inferred using the maximum likelihood method and the general time reversible model. The tree with the highest log likelihood (−17,206.33) is presented. Numbers at internal nodes represent the bootstrap support values (percentages) determined for 1000 replications (values < 50 are not shown). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 18 nucleotide sequences; the final dataset comprised a total of 1537 positions. Evolutionary analyses were conducted in MEGA11 [26]. The Brazilian bovine papillomavirus strain sequenced in this study is represented by a black-filled circle. Representatives of the viral PV types belonging to the seven species of the genus Deltapapillomavirus, recognized by the International Committee on Taxonomy of Viruses (ICTV), are shown. The GenBank accession numbers of the PV types included in the phylogenetic analysis are indicated.