The investigation, conducted by researchers at the Laboratory of Animal Virology and the Laboratory of Animal Pathology of the Universidade Estadual de Londrina in Paraná State, was published in the journal Frontiers in Virology. The team collected 23 hyperproliferative lesions from the esophagus of 16 asymptomatic mature Girolando breed dairy cows during post-slaughter inspection of the mucosa lining the whole upper gastrointestinal tract at a State-inspected meat processing plant. The animals came from dairy farms in the North Central mesoregion of Paraná, a region characterized by acid soil and bracken fern infestation, conditions long associated with papillomavirus-driven cancers of the bovine alimentary canal. Portions of each lesion were fixed in 10% neutral-buffered formalin for histopathological evaluation, while the remaining tumor fragments were stored at −80 °C for molecular analysis under an animal-use protocol approved by the university’s ethics committee.

Histopathological examination showed that all 23 mucosal lesions shared similar features consistent with a diagnosis of squamous papilloma. The esophageal tumors displayed a well-organized proliferation of stratified squamous epithelial cells arranged in multiple layers and forming papillary projections. Within the stratum spinosum of the proliferating epithelium, the pathologists occasionally observed small clusters of koilocytes, the hallmark cytopathic effect of papillomavirus infection, alongside prominent desmosomal junctions between epithelial cells and marked parakeratotic hyperkeratosis. Lymphocytic infiltrates were present in three of the tumors. These histological characteristics, which also included acanthosis and hypergranulosis in comparable lesions described in the literature, strongly suggested BPV infection, yet only five of the 23 lesions yielded viral DNA amplicons in the polymerase chain reaction assays employed.

To characterize the viral types present in the esophageal tumors, the researchers purified total DNA from all 23 frozen fragments using a commercial tissue kit and tested three consensual primer pairs designed to amplify conserved regions of the papillomavirus genome: FAP59/FAP64 and MY11/MY09, which target the L1 gene, and E1F2/E1R4, which targets the E1 gene. The L1 gene encodes the major capsid protein and is the most conserved open reading frame in the papillomavirus genome, serving as the reference for official type classification, while the E1 gene encodes a protein essential for viral replication. As an internal control to verify DNA integrity and exclude the presence of PCR inhibitors, the team amplified a 626-base-pair fragment of the ND5 gene of bovine mitochondrial DNA in every sample, and this control was successful in all lesions, including those that tested negative for BPV.

BPV DNA was detected in 21.7% of the esophageal lesions, five out of 23, and direct Sanger sequencing of the amplified partial L1 and E1 gene fragments revealed a striking diversity of viral types. Lesion A1 contained BPV9, with 100% nucleotide similarity in the L1 FAP amplicon to a reference sequence and 99.81% similarity in the E1 gene to another strain of the same type. Lesion A6 yielded BPV40, showing 97.25% similarity in the FAP sequence with a Brazilian BPV40 strain and 95.35% similarity in the partial E1 gene. Lesion A14 contained BPV4, the viral type most classically associated with tumors of the bovine upper gastrointestinal tract, with similarities of 99.11% and 99.59% in the L1 and E1 genes respectively. Lesion A16 harbored BPV1, a Deltapapillomavirus, with near 100% identity in both genes. Notably, BPV9 and BPV40 had never previously been detected in mucosal lesions of the bovine upper gastrointestinal tract, making this the first report of these types at that anatomical site.

The most remarkable finding came from lesion A7, which contained DNA from a putative novel BPV type. The partial L1 gene fragment amplified with the FAP59/FAP64 primer pair showed only 77.05% nucleotide similarity with BPV12, the closest previously characterized type, a divergence far exceeding the 10% threshold that officially defines a new papillomavirus type. To investigate further, the researchers amplified the viral genome from this lesion using multiply-primed rolling-circle amplification, a sequence-independent technique optimized for circular DNA virus genomes that generates multiple tandem copies of the full-length viral DNA, visible as a characteristic high-molecular-weight band on agarose gel electrophoresis. A primer-walking strategy with four additional sequencing primers then allowed the team to determine the complete nucleotide sequence of the L1 open reading frame of the isolate, which they named BPVnewXi_ESP/L1/BRA/UEL-Animal_7.

The complete L1 gene sequence of the putative novel type showed 75.18% nucleotide similarity with the L1 gene of a BPV12 strain previously characterized from a teat papilloma collected in 2016 from a dairy cow in Rio Grande do Sul state, Brazil. Phylogenetic reconstruction based on complete L1 nucleotide sequences of all BPV types already characterized through complete genome sequencing, representing the genera Deltapapillomavirus, Epsilonpapillomavirus, Xipapillomavirus, Dyoxipapillomavirus, and Dyokappapapillomavirus, placed the novel isolate in a well-supported clade together with BPV types 12, 30, 41, and 42, indicating that the strain probably belongs to the Xipapillomavirus 2 viral species. Because the genome of this isolate has so far been only partially characterized, through the complete L1 gene sequence, it can only be officially considered a new BPV type once its complete viral genome has been sequenced, according to current papillomavirus classification criteria.

The study’s authors emphasize that the diversity of BPV types associated with esophageal papillomas in dairy cattle is greater than indicated by initial studies, which focused almost exclusively on BPV4. BPV4, first isolated in 1978, has been confirmed as the dominant type in alimentary tract tumors across several geographic regions: in situ hybridization detected BPV4 DNA in 61 of 70 lesions from Glasgow slaughterhouses, molecular analysis identified BPV4 in papillomatosis extending from the mouth and tongue to the reticulum in animals from Scotland and England, and in Italy BPV4 was confirmed in more than 60% of esophageal papillomas detected among 1,133 slaughtered cattle. More recently, investigations of ruminal papillomatous lesions from northern Brazil detected BPV13, BPV44, and BPV2, and a study from India found BPV1 and BPV5 in the upper gastrointestinal tract of cattle and buffaloes. The esophageal types found in Paraná differed from those previously reported in the ruminal mucosa of northern Brazil, underscoring the need for in-depth investigations of papillomaviral diversity in the digestive mucosa of cattle raised in different geographic areas.

Interestingly, BPV2, a Deltapapillomavirus commonly associated with esophageal fibropapillomas and with mixed infections alongside BPV4, including a recently documented mixed BPV2 and BPV4 infection in a carcinoma in situ of the bovine esophagus from southern Brazil, was not amplified from any lesion in the present study. The authors note that the occurrence of mixed BPV infections may have been overlooked, since previous comparisons of PCR systems have shown that type-specific primers are more sensitive than consensual primers for detecting mixed infections, and mismatches between consensual primers and BPV genomic sequences can lower their sensitivity. On the other hand, consensual primers have repeatedly proven effective at uncovering previously uncharacterized BPV types, including putative new types and subtypes from cutaneous papillomas of Brazilian dairy cattle, and the present study demonstrates that combining multiple consensual PCR assays enhances detection sensitivity and enables the identification of additional viral types not previously observed in the bovine upper gastrointestinal tract.

The findings carry implications beyond veterinary pathology. Papillomaviruses are oncogenic viruses whose study in cattle has historically informed understanding of virus-driven carcinogenesis in humans, and the interaction between BPV and bracken fern compounds in the bovine alimentary canal remains one of the best natural models of environmental carcinogen and virus synergy. The Paraná study suggests 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, and that novel and previously unreported viral types may be present in mucosal lesions. The original contributions of the study, including the partial L1 and E1 gene sequences of the five detected BPV types, are publicly available in the NCBI GenBank database under accession numbers PZ565303 through PZ565312, providing a resource for future efforts to fully characterize the putative novel Xipapillomavirus and to map the true papillomaviral diversity of the bovine digestive tract.

Subject of Research: Virology

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

Article References: Matias, B. F., Lunardi, M., Cesário, G. D., Bracarense, A. P. F. R. L., Alfieri, A. F., & Alfieri, A. A. (2026). Multiple bovine papillomavirus types in squamous papillomas of the upper gastrointestinal tract of dairy cows from southern Brazil. Frontiers in Virology, 6, Article 1871673. https://doi.org/10.3389/fviro.2026.1871673

Image Credits: AI Generated

DOI: 10.3389/fviro.2026.1871673

Keywords: bovine papillomavirus, BPV, dairy cattle, esophagus, squamous papilloma, Xipapillomavirus, bracken fern, Brazil, L1 gene, E1 gene, Multiple, bovine