Case Report: Canine papillomavirus type 8 and 9 co-infection in pigmented viral plaques concurrent with squamous cell carcinoma in a Chinese crested dog

Introduction

Papillomaviruses (PVs) are small, circular, double-stranded DNA viruses known to induce a wide spectrum of epithelial proliferative disorders, ranging from benign hyperplasias to malignant neoplasms, across diverse vertebrate hosts including humans, cats, horses, dogs, and fish (1). In canine species, PV infections are predominantly subclinical; however, certain viral types have been implicated in the development of both hyperplastic and, more rarely, neoplastic lesions (2). To date, 33 distinct canine papillomavirus (CPV) types have been identified and taxonomically classified into three genera—Lambdapapillomavirus, Taupapillomavirus, and Chipapillomavirus—as catalogued by the Papillomavirus Episteme (PaVE) database (https://pave.niaid.nih.gov), although several types remain unclassified.

CPVs have been consistently associated with a range of epithelial lesions, including oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs) (2–8, p. 202; 9). Although pigmented plaques in dogs are generally small and confined to localized regions, there are documented cases in which these lesions present extensively and disseminate across broader cutaneous surfaces. In a subset of such cases, progression to SCC has been observed, often involving distinct CPV genotypes associated with malignant transformation (2, 4, 5, 10). The mechanisms underlying this malignant transformation remain incompletely understood, but viral genome oncogenes presence and, especially, host factors are thought to play contributory roles (2, 3).

The phenomenon of viral co-infection—well-documented in both human and veterinary medicine—has garnered increasing attention due to its potential to modulate disease severity, clinical presentation, and oncogenic potential (11, 12). Co-infection with multiple papillomavirus types has been associated with increased lesion severity and may influence carcinogenesis through complex virus-host interactions (13). In humans, PV co-infections have been strongly associated with increased severity and dissemination of cervical cancer (11, 13, 14). Despite these insights, reports of CPV co-infection in dogs remain exceedingly rare (14), and their clinical implications are poorly characterized.

Advancements in molecular diagnostics have significantly enhanced the detection and characterization of PVs. Multiply primed rolling circle amplification (RCA), particularly when coupled with high-throughput sequencing (HTS), has emerged as a robust methodology for the identification of circular viral genomes, enabling the discovery of novel PVs and the detection of co-infections across multiple species (15–17). This approach offers distinct advantages over conventional PCR techniques, which rely on degenerate primers and may fail to amplify divergent or co-infecting PV types. Moreover, the limitations of traditional culture systems—such as the need for complex raft cultures to obtain purified viral DNA—further underscore the utility of RCA-HTS in PV research (18).

Therefore, the aim of the present study was to characterize the PVs associated with extensive pigmented viral plaques in a Chinese Crested dog using rolling circle amplification RCA and HTS techniques, while confirming PV presence in the concurrent SCC by conventional PCR. Additionally, this study underscores the importance of investigating the molecular dynamics of CPV co-infections and their potential role in the pathogenesis and malignant progression of canine cutaneous lesions.

Case description

A 10-year-old, intact male Chinese Crested dog was referred to a veterinary dermatology service in Porto Alegre, Brazil, for evaluation of cutaneous lesions that had been slowly progressing over the past five years. Previous treatments reported by the owner, with limited success, included oral and injectable corticosteroids, monthly oral afoxolaner, and topical moisturizers and shampoos. Hematologic and biochemical analyses were unremarkable, and the dog was otherwise healthy.

Physical examination revealed multiple cutaneous lesions affecting different anatomical regions. On the naturally darker skin of the trunk and limbs, multiple pigmented plaques with black and yellow coloration were observed, ranging from flat to raised lesions (Figures 1A, B). On the lighter skin of the abdomen and neck, erythematous plaques covered by yellowish crusts were also present. Generalized comedones, skin dryness, and scaling were additionally noted. At the time of presentation, ulcerated lesions were also identified, including a large, ulcerated lesion on the right lateral aspect of the neck (Figure 1C) and two smaller ulcerated lesions located on the right and left flanks (Figure 1A).

The dog subsequently underwent skin biopsy under general anesthesia. Tissue samples were collected from multiple pigmented plaques (Sample 1), from the ulcerated lesion on the neck (Sample 2), and from one ulcerated lesion on the flank (Sample 3). Histopathological examination diagnosed Sample 1 as canine pigmented viral plaque (Figure 2), Sample 2 as a moderately well-differentiated squamous cell carcinoma (Figure 3), and Sample 3 as carcinoma in situ. A fragment from Sample 1 was stored at −20 °C for subsequent molecular analyses, whereas molecular analysis of Sample 2 was performed using DNA extracted from the paraffin-embedded tissue block. Although it was not possible to confirm whether the SCC (Sample 2) originated from a pre-existing plaque, it developed in a previously affected cutaneous region.

Based on these findings, the patient underwent a second surgical procedure during which the ulcerated lesions were excised. Topical treatment with imiquimod 5% cream was then initiated for the remaining plaque lesions and applied once daily. After 21 days, the patient exhibited an excellent clinical response, with complete resolution or flattening of most plaques. However, adverse effects were observed, including skin depigmentation and a focal inflammatory lesion on the right flank characterized by erythema, swelling, exudation, and pruritus, suggestive of a local burn reaction. Treatment was temporarily discontinued, resulting in resolution of the lesion. Imiquimod therapy was subsequently reinitiated at a reduced frequency of twice weekly.

Fourteen months after the initial presentation and following seven months of twice-weekly imiquimod treatment, the dog maintained good control of most plaque-like lesions, with only a few remaining raised. However, a new ulcerated lesion developed on the right side of the body (Figure 1D). Histopathological examination of this lesion confirmed carcinoma in situ. This later lesion was not available for molecular investigation.

Diagnostic assessment

Histopathology

For histopathological evaluation, tissue samples were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with H&E, and, when indicated, subjected to periodic acid–Schiff (PAS c/d) staining, following standard protocols in veterinary pathology. The analysis of Sample 1 (Figure 2), obtained from pigmented plaques, revealed an endophytic scalloped proliferation of spinous layer keratinocytes, accompanied by hypergranulosis, hyperpigmentation, and orthokeratosis in a basket-weave pattern. The underlying dermis exhibited vascular congestion, mild edema, and a discrete perivascular inflammatory infiltrate, with no pathological changes observed in hair follicles, sebaceous glands, or apocrine glands. PAS staining was negative, and the findings were consistent with a diagnosis of canine pigmented viral plaque. In contrast, Sample 2 (Figure 3), collected from the ulcerated lesion on the neck, demonstrated epidermal ulceration associated with a nodular infiltrative transformation characterized by poorly defined borders and absence of a fibrous capsule. Large polygonal tumor cells, morphologically resembling spinous layer keratinocytes, infiltrated the dermis and formed trabeculae and compact, coalescent cell blocks, with multifocal dyskeratosis and keratin pearl formation. Cellular atypia was evident, with anisokaryosis, anisocytosis, nuclear irregularities, prominent nucleoli, and aberrant mitotic figures, yielding a mitotic index of one mitosis per 40× field. Altogether, these features supported the diagnosis of a moderately well-differentiated squamous cell carcinoma.

HTS and sequencing analysis

Pigmented plaque (Sample 1) was macerated, and PBS was added to the pool for a final volume of 10 mL for subsequent low speed centrifugation - 2000×g for 30 min at 10 °C. The supernatant passed through a 0.45 μm filter. The nucleic acids were extracted with the commercial MagMAX^™ CORE nucleic acid purification kit (Applied Biossystems, Waltham, Massachusetts, USA), using the automated KingFisher^™ Duo Prime equipment (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). To sequence the papillomavirus whole genome, we proceeded with multiply primed RCA to enrich the sample followed by HTS as previously described¹⁸. The shotgun metagenomic library was performed using the Illumina DNA Prep kit, according to the manufacturer’s recommendations, and the sequencing was performed on the Illumina Miseq platform using the 600 cycle V3 reagent kit (Illumina Inc., San Diego, California, USA).

The HTS generated 4,361,942 paired-end high-quality reads, 3,940,448 reads classified as Chipappilomavirus 1 (CPV9) and 409,298 as Chipappilomavirus 3 (CPV8) through CZID online tool, revealing two complete and distinct circular contigs. Raw sequencing reads were initially evaluated using FastQC to assess overall sequencing quality which demonstrated more than 95% of bases presenting Phred quality scores above Q30. Low-quality bases and residual adapter sequences were trimmed (Trimmomatic software) prior to assembly, and only high-quality reads were retained for downstream analyses. De novo assembly was performed using SPAdes, and the resulting contigs were subsequently analyzed and manually inspected in Geneious Prime software through read mapping and coverage validation. Circular genome organization was confirmed by overlapping terminal regions and consistent read mapping across the complete genomes. A negative control was included throughout the sequencing workflow and yielded no papillomavirus-related reads. In addition, no other papillomavirus-positive samples were processed simultaneously. The identification of two distinct papillomavirus genomes was further supported by the high nucleotide divergence observed between CPV8 and CPV9 reference sequences, together with the independent assembly of two complete circular genomes exhibiting consistently high sequencing depth and coverage for both viruses.

The contigs formed two complete genomes of CPV9 and CPV8 (named as CPV9_LMM and CPV8_LMM respectively) with 8,396 bp and 7,953 bp, respectively. Each genome contains the typical papillomavirus gene set, including the early genes, which are involved in viral replication and cell transformation, and the late genes, which encode structural proteins essential for virion assembly (Figures 4A, B). The genomes also feature a non-coding upstream regulatory region (URR) that controls gene expression. High sequencing depth, with 20,509X and 4,412X of average coverage respectively, ensured reliable identification of all genomic regions. The phylogenetic tree was reconstructed with optimized alignments based on the nucleotide sequence of the L1 gene using Bayesian analysis [23]. The genomes CPV9_LMM and CPV8_LMM presented 99.64% and 99.85% of nucleotide identity with Canine papillomavirus 9 (NC_016074.1) and Canine papillomavirus 8 (NC_016014.1) complete genome reference sequences respectively. Phylogenetic reconstruction based on complete L1 gene reinforced the previous nucleotide analysis and can be observed in Figure 4C.

Conventional PCR papillomavirus assay in squamous cell carcinoma

The DNA was also extracted from the paraffin-embedded SCC (Sample 2 - ulcerated lesion on the neck). The tissue was suspended in 100 μL of digestion buffer (comprising 90 μL of 50 mM Tris, pH 8.0, and 10 μL of 20 mg/mL proteinase K). After an overnight incubation at 37 °C, the proteinase K was inactivated at 95 °C for 10 min, and the sample was centrifuged at 14,000 rpm for 2 min. Subsequently, 90 μL of the supernatant was transferred to a new tube, and the total genomic DNA was purified using the MagMAX™ Core Nucleic Acid Purification Kit, following the manufacturer’s instructions. The partial L1 gene was amplified from the extracted DNA using FAP59/FAP64 primer pairs in a standard polymerase chain reaction (PCR) (19). The resulting PCR products were electrophoresed on a 2% agarose gel for analysis. The positive sample was purified and subsequently sequenced bidirectionally using the same primers on an ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Based on this analysis, the paraffin−embedded SCC sample (Sample 2) tested positive for viral DNA. Amplification of the partial L1 gene by PCR using FAP59/FAP64 primers produced a specific product, which was subsequently subjected to bidirectional Sanger sequencing. The resulting sequence was identified as CPV8, showing 100% nucleotide identity with the CPV8 sequences previously obtained from the pigmented plaques.

Discussion and conclusion

Pigmented plaques are a well-recognized dermatological condition in dogs, most commonly affecting pugs and miniature schnauzers (2, 7, 9). Although they have also been documented in other breeds, to the authors’ knowledge, pigmented plaques have not previously been reported in a Chinese Crested dog. These lesions are frequently associated with immunocompromised state; however, it has also been described in immunocompetent individuals (3), as observed in the present study. In most cases, pigmented viral plaques tend to remain multiple but localized, with predominant involvement of the head, trunk and limbs (7). However, disseminated diseases, as observed in the dog described here, have been rarely reported.

Histopathological evaluation of the pigmented plaques confirmed the diagnosis of canine pigmented viral plaque, and next−generation sequencing (NGS) enabled the complete genome characterization of both CPV8 and CPV9. In parallel, the ulcerated lesion on the neck was confirmed by histopathology as a moderately well−differentiated squamous cell carcinoma. Although NGS could not be performed on the SCC due to financial constraints, viral DNA was successfully extracted from this paraffin−embedded tissue and analyzed by conventional PCR followed by Sanger sequencing, which identified only in CPV8. This finding reflects a methodological limitation; however, the detected CPV8 sequence showed 100% nucleotide identity to the CPV8 genome previously obtained from the viral pigmented plaque through NSG, demonstrating that the same strain present in the viral plaque was also detected in the SCC, which could suggest a possible prior progression from a viral plaque to the SCC associated with PV infection.

The malignant potential of these documented CPVs remains a subject of hypothesis, and the involvement of other PV types in the development of viral plaques cannot be ruled out. Although viral plaques are generally regarded as benign lesions, rare cases of neoplastic transformation into SCC with visceral metastasis have been reported (3–5). In dogs, PV-induced pigmented plaques and papilloma can undergo malignant transformation, but this phenomenon is uncommon, and most SCCs do not originate from PV-associated precursor lesions. In the reported case, papillomaviruses were identified both in the pigmented plaque and in the SCC, and CPV8 was consistently detected in both lesions using different methodologies, supporting an association between CPV8 detection and SCC in this case.

One notable exception involves research dogs with a mutation in the common gamma chain, which develop papillomas that progress to metastatic SCC. In contrast to these rare scenarios, the present case suggests the progression from pigmented viral plaques to SCC, as confirmed by histopathological examination of multiple cutaneous lesions, which revealed, in the same dog, the diagnosis of canine pigmented viral plaques, squamous cell carcinoma and carcinoma in situ. These findings suggest the evidence of malignant transformation in the absence of known immunodeficiency or genetic predisposition, reinforcing the need to reconsider the oncogenic potential of CPVs in naturally occurring cases and to investigate host-virus interactions that may contribute to neoplastic progression.

The phenomenon of co-infection, as evidenced in this case, adds a significant layer of complexity to our understanding of the pathogenicity of canine papillomaviruses (CPVs). Co-infections involving multiple PV types may exacerbate the severity of lesions and increase the risk of malignant transformation, as happened in humans (11, 12, 14), primarily due to potential interactions between viral types (20, 21). Although instances of canine PV co-infections have been reported sporadically, this study presents the first documented evidence of concurrent infection with CPV8 and CPV9 in a pigmented plaque evolving to SCC. Notably, the genomic sequences obtained in this study revealed the presence of E6 and E7 oncogenes in both CPV8 and CPV9 genomes. The E6 and E7 genes are recognized as important factors in papillomavirus biology, as they lead to the synthesis of proteins that drive the continuous replication of infected epithelial cells and the associated papillomavirus DNA (7, 20, 22). As these infected cells migrate toward the upper layers of the epithelium, they start to express L1 and L2 capsid proteins, which are essential for viral assembly. Ultimately, as the epithelial cells undergo desquamation, fully assembled viral particles are released into the environment, thereby facilitating further transmission of (13, 23). This intricate cycle of viral replication and dissemination emphasizes the need for enhanced surveillance and research into the dynamics of CPV infections, particularly in the context of co-infections.

Genomic investigations have implicated multiple CPV types not only in PVPs, but also in a spectrum of neoplastic conditions, including SCC, basal cell carcinoma (BCC), and trichoblastoma (24, p. 202; 6, 7, 25). Pigmented plaques are uncommon dermatological lesions in dogs, typically associated with closely related Chipapillomavirus types. To date, CPVs identified in such plaques include CPV3, -4, -5, -8, -9, -10, -11, -12, -14, -16, -18, and -24 (3, 7, 8, 24–26).

In the present case, HTS of a pigmented plaque revealed a co−infection involving CPV8 and CPV9, reinforcing the notion of complex viral interactions in cutaneous lesions. Conventional PCR performed on the SCC sample detected only CPV8. This discrepancy is attributed to primer complementarity bias, as consensus primers used in conventional PCR may preferentially amplify one viral genome over another when co−infections are present. HTS, by contrast, enabled unbiased recovery of both complete viral genomes, underscoring its superior sensitivity and resolution in detecting mixed infections. These findings expand the known diversity of CPVs involved in canine dermatopathies and highlight the importance of advanced molecular techniques to accurately characterize viral co−infections and their potential role in lesion progression. A limitation of this study is the absence of tissue localization methods (ISH or IHC), which could strengthen the evidence. Thus, we acknowledge that CPV DNA detected in SCC may originate from superficial epithelium or adjacent viral plaques rather than tumor cells themselves.

Notably, this is the first documented case of CPV8/CPV9 co-infection detected in pigmented plaques concurrent with SCC development in a dog without any clinical or laboratory evidence of immunodeficiency, underscoring the potential for disseminated plaques with pronounced histological alterations to exhibit increased susceptibility to malignant transformation.

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Footnotes

Edited by: Pawel Zmora, Polish Academy of Sciences, Poland

Reviewed by: İlker Şahinkesen, Ankara University, Türkiye

Nomeda Juodžiukyniene, Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Lithuania