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

What makes the discovery extraordinary is the humble nature of the evidence. The species was not identified from a complete skeleton, a horn core, or any diagnostic morphology at all, but from small bone fragments that could not be assigned to any known animal by sight. This is a remarkable discovery of a large extinct species based on small non-diagnostic bones, says Love Dalén, senior author of the study and Professor of Evolutionary Genomics at the Centre for Palaeogenetics. Only through the extraction and sequencing of ancient DNA preserved within the fragments could the team determine that the bones belonged to an animal unknown to science.

The remains were recovered from Denisova Cave and other sites in southern Siberia, a region that has become one of the most important windows into the Ice Age world. By analysing ancient DNA from the unidentifiable fragments, the researchers succeeded in generating twenty ancient genomes spanning an astonishing 200,000 years of evolutionary time. Datasets of this temporal depth are exceptionally rare in ancient DNA research, and they allow scientists to track how populations changed across entire glacial cycles rather than merely sampling a single moment in the past. When the team compared these genomes with those of other bovine species, the result was unambiguous: the fossils belonged to a lineage entirely distinct from any bovine described to date.

The genomic comparisons allowed the researchers to estimate when this mysterious lineage split from its closest living relatives. According to their analysis, the species began diverging from modern yaks around 400,000 years ago and became fully separated around 250,000 years ago. A useful comparison for when this lineage originated is the divergence between brown bears and polar bears, which has also been estimated to have occurred around 400,000 years ago, says Alexandre Gilardet, one of the lead authors and a PhD student at the Centre for Palaeogenetics. This age overlaps with a particularly strong warm climatic period, which could have prompted population isolation and, ultimately, speciation. In other words, a prolonged warm interval may have fragmented ancestral yak populations, cutting groups off from one another long enough for them to evolve into separate species.

The parallel with another landmark discovery from the very same landscape is striking. Denisova Cave is already world-famous as the site where, in 2010, DNA analysis of a single finger bone fragment revealed the Denisovans, an entire group of archaic humans previously unknown to science. These findings mirror the discovery of the Denisovans, a group of archaic humans, from a single fossil in 2010 and highlights the Altai region as a possible biodiversity hotspot and refugium across Ice Ages, says Dalén. The idea that the Altai Mountains and surrounding areas served as a refugium, a place where populations survived through climatic extremes and diversified in isolation, gains considerable weight from the fact that two entirely new branches of the tree of life, one human and one bovine, have now been identified from the same region largely through genetic rather than anatomical evidence.

Yet the new bovine species appears to have had a very different evolutionary history from the Denisovans in at least one crucial respect. When different groups of archaic humans met in Eurasia, they frequently interbred, leaving traces of hybridization in the genomes of their descendants, including present-day people. In contrast, the scientists detected negligible evidence for interbreeding between the new species and other bovines living in the area at the time, such as the steppe bison or the aurochs, the extinct wild ancestor of domestic cattle. This reproductive isolation reinforces the conclusion that the animal was a genuinely separate species rather than a regional population or hybrid lineage, and it suggests that biological boundaries between these large Ice Age herbivores remained firmly in place even when their ranges overlapped.

The deep genomic time series also captured dramatic changes within the species itself. The results suggested that a population replacement occurred during the last interglacial warm period, accompanied by a loss of genetic diversity. This could reflect the genetic consequences of climate warming around 120,000 years ago on a cold-adapted species, says Jonas Oppenheimer, the other lead author of the study and a postdoctoral researcher at the Centre for Palaeogenetics. The long time span of our dataset is really rare in ancient DNA, and allows us to look across whole glacial cycles to see how they might have impacted biodiversity. The last interglacial, a period when global temperatures were warmer than today in many regions, appears to have squeezed cold-adapted fauna into smaller refugia, eroding their genetic variation and reshuffling populations in ways that left permanent signatures in their DNA.

The timeline of the species’ disappearance remains only loosely constrained. The youngest specimen analysed in the study is around 27,000 years old, placing the animal squarely within the last Ice Age, at a time when mammoths, woolly rhinoceroses, steppe bison and other megafauna still dominated the Siberian landscape. The species became extinct sometime after that point, but the exact timing and the causes of its demise remain unknown. Whether it succumbed to rapid climate warming at the end of the Pleistocene, to the spread of humans through the region, to competition with other large herbivores, or to some combination of these pressures is a question that future discoveries may help to answer.

Geographically, the extinct relative of modern-day yaks was detected at Denisova Cave and in the broader Altai-Sayan Mountains and southern Siberian region. The contrast with its living relatives is telling: modern wild yaks are today restricted to the high elevations of the Tibetan Plateau, where they survive in some of the most extreme cold environments on Earth. The new findings suggest that yak-like bovines once occupied a much broader distribution across mid-latitude Asia, and that southern Siberia harboured a distinct lineage that has since vanished without leaving any obvious trace in the fossil record as traditionally studied.

A formal species name, however, must wait. Genetic evidence alone is not sufficient to formally classify and name a new species under the rules of zoological nomenclature, which require comparison with diagnostic physical material. Going forward, the researchers therefore plan to compare their genomic findings with previously described yak-like remains from the region, in the hope of determining whether any of those fossils belong to the same species. If a match is found, the scattered bones in museum collections could suddenly acquire a new identity, and the ghost bovine of the Altai could finally receive both a name and a face. For now, the discovery stands as a powerful demonstration of how ancient DNA can reveal entire species hidden inside fragments too small and too degraded for traditional identification, and as a reminder that even the best-studied Ice Age ecosystems still hold surprises waiting in the sediment.

Subject of Research: Ancient DNA analysis of Ice Age bovine remains from Siberia revealing an extinct yak-relative species

Article Title: Previously unknown extinct bovine species discovered in Siberia

Article References: Previously unknown extinct bovine species discovered in Siberia. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: ancient DNA, bovine, yak, Siberia, Denisova Cave, Ice Age, extinction, speciation, Altai Mountains, palaeogenetics, Current Biology, megafauna