Molecular and Histological Identification of Bovine Papillomavirus 1, 2 and a Novel Genotype in Cutaneous Papillomas of Dairy Cattle in Taiwan

Abstract

Bovine papillomaviruses (BPVs) are host-specific and strongly epitheliotropic infectious agents that cause benign epithelial and mucosal proliferations, with potential for malignant transformation. However, BPV1, BPV2, and BPV5 are unique in their ability to infect both epithelial and connective tissues. While BPV infections had been documented globally, there was no disease information reported from Taiwan. To investigate whether BPVs are associated with the development of cutaneous papillomas in dairy cattle in Taiwan, in the present study, eight cutaneous papilloma samples from six dairy farms were collected and analyzed by using histopathology, immunohistochemical (IHC) staining, and molecular biology methods. BPV1 and BPV2 were identified, along with a novel BPV sharing 80.9% sequence identity with BPV38. This novel BPV, classified under Xipapillomavirus, was detected in both epithelial and mesenchymal cells through in situ hybridization (ISH), suggesting a broader tissue tropism than typical Xipapillomavirus infections. These findings provide new insights into BPV diversity and pathogenesis.

Keywords: Xipapillomavirus; bovine papillomavirus (BPV); cattle; fibropapilloma; in situ hybridization (ISH).

Figure 1

Histopathological findings from seven skin papillomas. The tissue slides were stained with hematoxylin and eosin (H&E). Fibropapillomas (Case nos. 23-00, -01, -03, -05, and -07) and papillomas (Case nos. 23-02 and -06) were diagnosed. Hyperkeratosis was noted in all the cases. Intranuclear inclusions were noticed in Case no. 23-00. Case no. 23-04 was submitted only for molecular detection thus being excluded.

Figure 2

Molecular diagnosis of PVs by two degenerative primers. The viral genome in each sample was amplified by CP4/5 (A) and MY9/11 (B). All the samples were positive for CP4/5, while only seven samples, except Case no. 23-02, were positive for MY9/11. L, ladder; Neg., negative control.

Figure 3

Detection of PVs by IHC staining. The BPV was detected by the anti-HPV antibody and the signals were visualized by the AEC chromogen system. Intranuclear red signals were considered true positive. In Case no. 23-00, the signals were strong in both keratinized epithelial cells and the underlying fibrous connective tissues. In Case nos. 23-01, -03, -05, and -07, only a few positive signals were detected on epithelial cells. In Case nos. 23-02 and -06, there was no signal.

Figure 4

Detection of novel BPV by ISH staining. To assess the presence of the viral genome in the lesion, ISH staining was performed. The positive signals were visualized by the DAB chromogen system and intranuclear brownish signals were considered true positives. Signals were detected mainly in the epithelium, whereas a small proportion of mesenchymal cells also had intranuclear positive signals. A negative control (Control) for excluding the melanin pigments and nonspecific signals was included.

Figure 5

Phylogenetic analysis of BPVs based on the L1 ORF. The full length of L1 ORF of 44 BPV genotypes was aligned using MEGA11 and a phylogenetic tree was generated using the maximum likelihood method based on the Tamura-Nei model. The accession numbers of each strain were labeled. According to the official updated information (https://pave.niaid.nih.gov/index), BPV30 appeared twice in the literature; thus, one was officially renamed BPV31, and the subsequent order was rearranged accordingly. Parentheses indicate the previous names for clarity. The BPVs identified in this study are marked with solid circles and the scale of branch length indicates genetic divergence.

Figure 6

Percentage of sequence identity of BPVs based on L1 ORF. The full length of L1 ORF of 44 BPV genotypes was aligned using MEGA11. All strains analyzed were identical to those used in the phylogenetic study. Sequence identity was calculated using DNAStar and the axial color gradient reflected the percentage of sequence identity between strains.