Multiple bovine papillomavirus types in squamous papillomas of the upper gastrointestinal tract of dairy cows from southern Brazil

Papillomavirus is an oncogenic virus that infects the cutaneous and mucosal epithelia of several host species. This study investigated the diversity of bovine papillomavirus (BPV) types in hyperplastic lesions of the esophageal mucosa in dairy cows from Paraná State, southern Brazil. A total of 23 hyperproliferative lesions were collected from the esophagus of 16 dairy cows and histologically classified as squamous papillomas. Consensual primer pairs FAP59/FAP64, MY11/MY09, and E1F2/E1R4 were used to partially amplify the L1 and E1 genes of BPVs. Amplicons were sequenced using the Sanger method. BPV DNA was detected in 21.7% (5/23) of the esophageal lesions. Sequence analysis identified BPV types 1 and 4, previously associated with such lesions, in two lesions. BPV types 9 and 40 were detected for the first time in mucosal lesions of the upper gastrointestinal tract (UGT) in two additional samples. Furthermore, one lesion contained DNA from a putative novel BPV type phylogenetically related to BPV12. Overall, five distinct BPV types were molecularly detected across five lesions. These findings suggest that, similar to cutaneous papillomatosis, multiple BPV types may be associated with papillomas in the esophageal mucosa of dairy cows, particularly those raised on bracken fern–contaminated pastures. The study highlights the importance of utilizing multiple consensual primer sets targeting conserved regions of the L1 and E1 genes to more accurately characterize BPV diversity in mucosal lesions, as novel and previously unreported viral types may be present.

Papillomaviruses (PVs) are oncogenic viruses that induce both benign and malignant lesions in the skin and mucosa of vertebrates, including humans (1). They are non-enveloped viruses with an icosahedral capsid whose genome is composed of a double-stranded circular DNA molecule of approximately 8 kbp (2). The PV genome contains open reading frames (ORFs) that encode early (E) and late (L) proteins. The E1 and E2 proteins promote viral replication, and the E4 protein participates in the amplification and production of virions. The oncoproteins E5, E6, and E7 are involved in the process of cellular transformation. The late-phase proteins L1 and L2 form the viral capsid and are required for the completion of the viral life cycle (3).

The L1 gene is considered the most conserved ORF within the viral genome. Therefore, to describe a new viral type, it is necessary to sequence the new genome to demonstrate that the nucleotide (nt) sequence of the L1 gene presents more than 10% divergence when compared to previously sequenced genomes already officially classified as viral types. If the new viral strain differs between 2 and 10%, a new subtype is considered, while a new variant is detected when the identity observed is <2% (4).

The bovine papillomavirus (BPV) types previously characterized in cattle belong to the genera Deltapapillomavirus (BPV1, 2, 13, and 14), Dyoxipapillomavirus (BPV7 and BPV-TAPV-lsx), Epsilonpapillomavirus (BPV5, 8, 25, and 44), Xipapillomavirus (BPV3, 4, 6, 9, 10, 11, 12, 15, 17, 20, 23, 24, 26, 28, 29, 30, 31, 35, 36, 37, 38, 39, 40, 41, 42, and 43), Dyokappapapillomavirus (BPV16, 18, and 22), however, some viral types remain unclassified (BPV19, 21, 27, 32, 33, 34, and 45) (5).

BPV infection may result in cutaneous papillomatosis, forming hyperplastic lesions on the skin of various anatomical sites, such as teats and udder. These lesions are characterized as squamous papillomas or fibropapillomas, considered benign (1). However, BPV infection may also result in malignancy, being the precursor of neoplastic lesions in the urinary bladder mucosa, generally associated with bracken fern (Pteridium spp.) consumption, known as bovine enzootic hematuria (BEH) (6–10). The bracken fern is known for its carcinogenic and immunosuppressive properties and may act synergistically with BPV, promoting malignancy (6).

Another clinical condition that can affect cattle and is associated with BPV infection is the development of tumors in the mucosa of the upper gastrointestinal tract (UGT), which is also related to bracken fern compounds. The viral types already detected in such lesions were BPVs 1, 2, 4, 5, 13, and 44 (8, 11, 12), with BPV4 being detected more frequently in both benign and malignant lesions (8). The cytopathic effects caused by BPV infection, often observed in such lesions, are koilocytosis, ortho and parakeratotic hyperkeratosis, hypergranulosis in the granular layer, and acanthosis (13). The main lesions observed in the alimentary tract are classified as papillomas, fibropapillomas, and squamous cell carcinomas (14, 15). This investigation aimed to histologically classify the hyperplastic lesions and investigate the diversity of BPV types in lesions from the alimentary tract mucosa of dairy cows raised in southern Brazil.

Biological samples and histopathological analysis

Through the post-slaughter inspection of the mucosa lining the whole UGT, 23 proliferative lesions were identified and collected from the esophagus of 16 asymptomatic Girolando breed mature cows from dairy farms located in the North Central mesoregion of the Paraná state, southern Brazil, a region known to contain acid soil and bracken fern infestation. A portion of each esophageal lesion was fixed by immersing in 10% neutral-buffered formalin solution, processed routinely for histopathological evaluation, and stained with hematoxylin and eosin. The proliferative mucosal lesions were classified by histopathology as previously described (13). The remaining fragments of each tumor were stored at −80 °C for further molecular analysis. The slaughter and sample collection were carried out in a State-inspected meat processing plant.

This study was approved by the Ethics Committee for Animal Use at the Universidade Estadual de Londrina (Protocol n° 050/2021). All applicable international, national, and institutional guidelines for animal care and use were followed.

PCR amplification of partial fragments of BPV L1 and E1 genes

All 23 frozen fragments from each esophageal lesion were disrupted and homogenized using TissueLyser LT (Qiagen, Hilden, Germany). Total DNA was then purified using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions, and stored at −80 °C for molecular assays. A cutaneous papilloma known to contain BPV type 6 was employed as positive control while ultrapure sterile water was used as a negative control in all DNA extraction procedures.

To investigate the presence of BPV potentially associated with the hyperplastic lesions of the bovine esophageal mucosa, three consensual primer pairs (FAP59/FAP64, MY09/MY11, and AR-E1F2/AR-E1R4), designed to amplify regions of the L1 and E1 conserved genes of a broad range of cutaneous and mucosal PV strains, were tested using DNA purified from the lesions. The PCR assays used to amplify fragments of the L1 and E1 genes, along with the sequences and characteristics of the primers, have been previously described (16).

To verify DNA integrity and to ensure the absence of PCR inhibitors, an endogenous internal control, a 626pb fragment of the ND5 gene present in the bovine mitochondrial DNA, was tested with BOV1 and 2 primers for each sample, following conditions previously described (9).

Direct sequencing of subgenomic fragments

Amplicons representing partial fragments of the BPV L1 and E1 genes were purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega, Madison, WI, USA), following the manufacturer’s protocol. Next, direct nt sequencing was performed on a 3500 Genetic Analyzer Sequencer (Applied Biosystems, Carlsbad, CA, USA), using the BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems, Carlsbad, CA, USA), and the corresponding forward and reverse primers used in the PCR assays, according to the instructions provided by the manufacturer. The nt sequences obtained were examined for quality analysis of chromatogram readings using the Phred application (http://lbi.cenargen.embrapa.br/phph/). Sequences with a minimum Phred quality score threshold of 20 (Q20) were considered acceptable. The consensus sequences were assembled using CAP3 software Version 3, and their identities were determined through comparison with nt sequences deposited in the public database GenBank, using the BLASTn program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Rolling-circle amplification of DNA from the putative new BPV type

The BPV genome was amplified using DNA purified from the esophageal lesion #A7 through the multiple-primed rolling-circle amplification (RCA) technique with the TempliPhi 100 amplification kit (Cytiva, Marlborough, MA, USA), following a previously reported protocol optimized for the amplification of papillomaviral genomes (17). The amplification of PV DNA was verified through electrophoretic analysis of the RCA product (2 μL) using a 0.8% agarose gel stained with ethidium bromide, generating a band corresponding to multiple tandem copies of the full-length PV DNA.

To obtain the complete nt sequence of the L1 gene of the herein named isolate BPVnewXi_ESP/L1/BRA/UEL-Animal_7, putative novel BPV type amplified from lesion #A7, a primer-walking sequencing strategy was adopted directly on the RCA product, with the use of four additional sequencing primers to cover the whole L1 gene (Table 1).

Phylogenetic analysis

Pairwise and multiple sequence alignments at the nt level, and sequence similarities were estimated using ClustalW in the MEGA v11 software (18). Complete nt sequences of the L1 gene derived from PV types, previously characterized through complete genome sequencing and representing the genera Deltapapillomavirus, Epsilonpapillomavirus, Xipapillomavirus, Dyoxipapillomavirus, and Dyokappapapillomavirus, as well as some viral types awaiting official classification, were included in the analysis. A phylogenetic tree was reconstructed based on the alignment of complete L1 nt sequences using the maximum likelihood method with the general time-reversible model in MEGA v11 software.

All 23 mucosal lesions exhibited similar histological features. The histopathological analyses of the neoplastic lesions located in the esophagus revealed a well-organized proliferation of stratified squamous epithelial cells (Figure 1). These cells were arranged in multiple layers and formed papillary projections (Figure 1A). Within the stratum spinosum of the proliferating epithelium, small clusters of koilocytes were occasionally observed (Figure 1B). Additionally, prominent desmosomal junctions were evident between epithelial cells (Figure 1C), while marked parakeratotic hyperkeratosis was noted (Figure 1D). These histological characteristics are consistent with a diagnosis of squamous papillomas. Furthermore, lymphocytic infiltrates were present in three of the tumors. Such findings are cytopathic effects that suggest BPV infection. However, only five of the hyperplastic lesions had partial fragments of the BPV L1 and E1 genes amplified by the PCR assays employed (Table 2), whereas DNA purified from all lesions had a partial fragment of ND5 gene of the bovine mitochondrial DNA amplified.

Five different types of BPV were amplified from hyperplastic lesions of the esophageal mucosa of cattle. Direct sequencing of the partial fragments amplified from L1 and E1 BPV genes revealed the presence of the previously characterized viral types BPVs 1, 4, 9, 40, and a putative new BPV type, which presented 77.05% nt similarity with the closest previously characterized BPV type 12 in the partial fragment of the L1 gene (FAP amplicon) (Table 2).

The direct sequencing of the partial BPV L1 gene amplified with the primer pair FAP59/FAP64 in sample #A1 revealed 100% similarity with the BPV9 corresponding sequence (GenBank accession number HM245433). The E1 gene partial sequence was also amplified in the same lesion. The direct sequencing of the E1 gene demonstrated 99.81% nt similarity with another strain of the same viral type (GenBank accession number MW436427). In sample #A6, Blastn analysis of the FAP sequence demonstrated 97.25% similarity with BPV40 strain BPV/UFPE03BR (GenBank accession number JQ897974) and 93.81% similarity with BPV40 complete genome (GenBank accession number MW727481). The sequence from the partial E1 gene showed 95.35% similarity with the BPV40 partial sequence (GenBank accession number MW428426). As for lesion #A14, nt sequences amplified from the L1 and the E1 genes demonstrated similarities of 99.11% and 99.59% with BPV4, respectively (GenBank accession number MW436424). In lesion #A16, nt sequences obtained from the L1 gene with primer pair MY09/MY11 and from the E1 gene demonstrated similarities near 100% with BPV1 (GenBank accession numbers MG263871 and MG977494, respectively).

Nonetheless, the nt sequence representing a partial fragment of the L1 gene (FAP59/FAP64) in tumor #A7 showed only 77.05% nt similarity with BPV12 (GenBank accession number OP682876), which indicates the identification of a putative new BPV type. Blastn analysis of the contig assembled from reads generated by primer-walking sequencing of the RCA product, obtained from the same BPV strain representing a putative new BPV type, revealed that the nt sequence encompassed the whole L1 ORF and showed 75.18% similarity with the corresponding L1 gene sequence of the BPV12 strain 3895RS16-BR node 4 (GenBank accession number MW436428). This BPV12 strain was previously characterized from a teat papilloma collected from a dairy cow raised in 2016 in a farm in Rio Grande do Sul state, Brazil.

Phylogenetic reconstruction based on the complete L1 nt sequences of all BPVs already characterized through the complete genome sequencing and classified in the Deltapapillomavirus, Epsilonpapillomavirus, Xipapillomavirus, Dyoxipapillomavirus, and Dyokappapapillomavirus genera, demonstrated that the putative novel BPV type herein identified as BPVnewXi_ESP/L1/BRA/UEL-Animal_7, and derived from an esophageal papilloma, grouped together in a well-supported clade with BPVs type 12, 30, 41, and 42, suggesting that this strain probably belongs to the Xipapillomavirus 2 viral species (Figure 2).

In this study, consensual primer pairs capable of amplifying partial fragments of the conserved PV L1 and E1 genes enabled the detection of BPV genomes in 5 out of 23 esophageal tumors of dairy cows. The direct sequencing of the obtained amplicons revealed the infection by representatives classified in the Deltapapillomavirus genus (BPV1) and the Xipapillomavirus genus (BPV4, BPV9, and BPV40), as well as a putative new BPV type most closely related to BPV12 (75.18% similarity), also to be classified in the Xipapillomavirus genus.

Furthermore, a partial fragment of the bovine mitochondrial DNA was successfully amplified from all esophageal papillomatous lesions, which excludes the presence of PCR inhibitors and assures DNA integrity in the negative samples. These findings may indicate the occurrence of false-negative results represented by the esophageal lesions that did not generate BPV amplicons in the PCR assays employed, especially due to the fact that histopathological findings consistent with squamous papilloma classification was present in all evaluated esophageal tumors, irrespective of the PCR results.

BPV4, isolated in 1978, is considered the main viral type associated with tumors that develop in the UGT of cattle (14, 19). Studies conducted in diverse geographic areas have confirmed this finding. The analysis of lesions collected from slaughterhouses in Glasgow, representing 70 different cases, detected the presence of BPV4 DNA using the in situ hybridization technique, and confirmed the infection by this viral type in 61 lesions (20). In another investigation involving papillomatosis extending from the mouth and tongue to the reticulum in two different animals from Scotland and England, the molecular analysis demonstrated the identification of the BPV4 genome in those lesions (21). In Italy, exophytic and inverted papillomas were detected by histopathological evaluation of the esophagus of 147 out of 1,133 cattle slaughtered between 2000 and 2001. Through molecular analysis, the presence of BPV4 was also confirmed in more than 60% of the tumors (8).

Through a molecular strategy combining assays employing three different consensual primer pairs (FAP59/FAP64, MY09/MY11, and AR-E1F2/AR-E1R4), able to amplify conserved genomic segments of the PV genome, our investigation revealed a broader diversity in BPV types than classically related to hyperplastic lesions that occur in this specific anatomical site. Although used by other authors, the MY09/MY11 primer pair did not demonstrate efficacy in the present study, as it amplified PV DNA in only one sample of esophageal lesion. To the authors’ knowledge, this study represents the first report that detected PV DNA from BPV9 and BPV40 in tumors located in the mucosa of the UGT of cows. Recently, a study investigating the presence of BPV DNA in 100 papillomatous lesions of the ruminal mucosa of cattle from northern Brazil, using PCR with the FAP primer pair, detected BPV13 and BPV44 in these tumors, in addition to BPV2, which is commonly associated with papillomas from bovine UGT (12).

Our findings, combined with the unusual BPV types previously detected in the rumen from cattle (BPV13 and 44), suggest that there is a greater diversity of viral types associated with tumors of the UGT of cattle than previously indicated by initial studies (8, 15). Interestingly, despite having detected the association of new viral types in the esophageal mucosa of cattle, the BPV types detected herein differed from the viral types previously found in the ruminal mucosa of cattle from northern Brazil, highlighting the need for in-depth investigations aiming to elucidate the papillomaviral diversity in digestive mucosa of cattle raised in different geographic areas.

Another viral type commonly found in lesions located at the UGT of cattle is the Deltapapillomavirus BPV2, which is usually associated with esophageal fibropapillomas (11, 15). Molecular analyses of hyperplastic lesions collected from this anatomical site from an affected bull raised in the midwestern region of Brazil demonstrated the occurrence of a mixed infection caused by BPV2 and 4 through sequencing of the L1 and E1 gene fragments amplified by FAP59/FAP64 and E1F2/E1R4 primer pairs (22). More recently, a mixed infection characterized by the detection of BPV2 and BPV4 was also confirmed in a carcinoma in situ of the esophagus of a bull from southern Brazil using rolling circle amplification followed by high-throughput sequencing (16). However, BPV2 DNA was not amplified from any lesions herein evaluated. In the current investigation, the occurrence of mixed BPV infections may have been overlooked. A previous study that compared PCR systems employing BPV type-specific primers with the PCR using FAP primers revealed that the former strategy was more sensitive and able to detect mixed infections when compared with the PCR assay with consensual primers. Mismatches between consensual primers and BPV genomic sequences may lower the sensitivity of these types of primers. On the other hand, numerous putative new BPV types were detected by using consensual primers in previous studies, which collaborated substantially to extend the knowledge of BPV diversity (3, 12, 23, 24).

This study also detected the BPV1 DNA in a lesion from the bovine esophagus, which was also identified in the UGT of buffaloes from India. In addition to this finding in buffaloes, this previous investigation detected the BPV5 in the same anatomical location of cattle from the same geographic area (11).

In addition to the detection of genomic sequences from BPV types previously characterized, the sequencing and phylogenetic analysis of the complete L1 gene amplified by RCA from the tumor #A7 evaluated in this study showed the presence of a putative new BPV type DNA closely related to the Xipapillomavirus BPV12 (75.18% identity), demonstrating its probable classification as a representative of the Xipapillomavirus 2 species (Figure 2). Regarding the current classification for new PVs, the sequencing and analysis of the identity observed in the nt sequence of the L1 ORF, the most conserved gene in the PV genome, define the classification of a new PV type, subtype, or variant. However, as the putative novel BPV type identified herein had its genome only partially characterized, through the complete L1 gene sequence, this BPV strain could only be officially considered a new BPV type, to be classified in the Xipapillomavirus genus, upon sequencing of its complete viral genome.

The use of consensual primers to amplify conserved regions of the BPV genome represents a powerful molecular approach for elucidating the genetic diversity of BPV types associated with hyperplastic lesions in cattle. Previously, the analyses of cutaneous papillomas through FAP59/FAP64 allowed the characterization of a putative new BPV type from a skin papilloma collected from a dairy cow raised in Brazil (25). The molecular investigation by FAP PCR assay of cutaneous warts from cattle raised in northern Brazil also enabled the identification of nine possible new BPV subtypes and four probable new BPV types in cattle affected by cutaneous papillomatosis (23). Collectively, these findings underscore the effectiveness of consensual primers in detecting previously uncharacterized BPV types. Consistent with this evidence, the present study further demonstrates that combining multiple consensual PCR assays enhances detection sensitivity, enabling the identification of additional viral types not previously observed in the bovine UGT.