IntroductionKeratinocytes are key barrier cells able to mount a robust interferon (IFN) antiviral response to defend against infection in the skin. Similar to humans, dogs spontaneously develop skin disorders associated with dysregulation of IFN immunity and can be used as a large animal model to investigate these diseases. One of the critical factors driving IFN regulation are interferon regulatory factors (IRFs). IRFs are crucial in upregulating the antiviral type I or type III IFNs, which then subsequently upregulate hundreds of antiviral effector proteins called interferon stimulated genes (ISGs).MethodsWe sought to comparatively analyze how IRF1, 3 and 7 regulate type I and type III IFNs and ISGs using canine and human keratinocyte cultures. To this aim, we stimulated keratinocytes in vitro with dsDNA and dsRNA ligands and analyzed upregulation of IFNs and ISGs using real time quantitative PCR (RT-qPCR). Knockout of IRF3 and IRF1 and knockdown of IRF7 was performed on dog and human keratinocytes using CRISPR-Cas9 and shRNA methods, respectively. Knockdown or knockout was confirmed using RT-qPCR and/or western blots.ResultsWe demonstrated that compared to canine keratinocytes, human keratinocytes express higher basal type I IFN-β and ISGs, induce higher levels of type III IFNs upon stimulation, and overall, express higher IFN and ISG copies. We further showed that IRF regulation of induced IFNs, particularly IRF1, can be species-specific and/or stimulus-specific, and putatively that non-IRF mechanisms likely mediate basal expression of type I IFNs in human keratinocytes and type III IFNs in canine keratinocytes. While both IRF3 and IRF7 are critical for type I and III IFN induction following activation of cytosolic dsDNA and dsRNA receptors, neither can account for differences in the induced type I versus III IFN expression between dog and human, suggesting non-IRF1, 3, or 7 mechanisms underly this regulation.ConclusionsThese studies provide support for use of the dog as a model to discern mechanisms underlying high basal type I and ISGs in human keratinocytes, how high basal ISGs can modulate the antiviral response, and uncover non-IRF mediated mechanisms regulating the type I versus type III response in keratinocytes.

Papillomas, many of which are virally induced, are common proliferative cutaneous and mucocutaneous lesions in multiple species, exhibiting characteristic histologic cytopathic changes that distinguish them from nonviral squamous papillomas. A single case report of a novel papillomavirus, Ursus maritimus papillomavirus-type 1, in a polar bear has been reported without investigation into any association between this virus and papilloma formation. We identified papillomas in 3 polar bears. All 3 cases had pedunculated masses consistent with papillomas (i.e., proliferative epithelium forming papillary projections on a fibrovascular stalk); case 1 also exhibited koilocytosis (cytopathic change), consistent with a viral papilloma. Polymerase chain reaction (PCR) using primers that can amplify a diversity of papillomaviruses followed by amplicon sequencing yielded a novel papillomavirus sequence in case 1, which shared <70% nucleotide identity to any known papillomavirus type, indicative of a putatively novel papillomavirus. In situ hybridization (ISH) of case 1 demonstrated viral nucleic acid within proliferative cells and not within the adjacent normal skin, suggesting the virus was the causative agent of this papilloma. The squamous papillomas in cases 2 and 3 were negative for papillomavirus by both PCR and ISH. These findings support our hypothesis that cytopathic effect is associated with the presence of papillomavirus in polar bears, while the lack of histologic cytopathic change may predict nonviral pathogenesis. Further sequencing of the putatively novel viral genome will benefit research and conservation efforts of polar bears.

Cutaneous plaques and squamous cell carcinoma (SCC) are common in captive North American snow leopards (SLs) ( Panthera uncia). Our objective was to determine whether these lesions are potentially associated with papillomavirus(es). Polymerase chain reaction (PCR) was performed on 3 cutaneous plaques using degenerate primers for papillomaviruses. A putatively novel papillomavirus was identified that shared 76% sequence identity to Felis catus papillomavirus 2. Specific PCR for this virus was performed on 5 cutaneous SCC samples and 7 normal skin samples, which were all positive. In situ hybridization for this putatively novel virus was performed, which revealed strong hybridization signals within hyperplastic cells in cutaneous plaques (n = 3) and within neoplastic cells in cutaneous SCC samples (n = 5). No hybridization signals were identified within normal skin. Ultimately, identification of a causal viral agent in the development of plaques and SCC in SLs will help guide therapeutic intervention and lay the foundation for development of prophylactic vaccines.

A claw bed inverted squamous papilloma (ISP) presented clinically as a swollen digit in a dog. Canine papillomavirus (CPV) type 2 was amplified by PCR and localised to the papilloma's epidermis using in situ hybridisation. This is the first report demonstrating a claw bed ISP caused by CPV.

The aetiology of oral squamous cell carcinoma (SCC) in horses is unknown, but papillomavirus infection as well as chronic periodontal disease are suspected to play a pathogenic role. In humans, some oropharyngeal cancers develop in association with human papillomaviruses. Equus caballus papillomavirus 2 (EcPV2) is suspected to play a causal role in the development of equine genital SCC. Given that association, we hypothesized that EcPV2 is associated with the development of oral SCC in horses. We performed standard polymerase chain reaction (PCR) and in-situ hybridization (ISH) for EcPV2 on 31 formalin-fixed paraffin-embedded equine oral SCCs (lingual, gingival, palate) and 10 equine non-SCC oral samples. PCR for EcPV2 was positive in 10/31 (32%) oral SCCs while all non-SCC oral samples were negative. Intense hybridization signals for EcPV2 nucleic acid were detected by ISH within neoplastic epithelial cells in 8/31 (26%) oral SCCs but not in the adjacent normal oral mucosa. No hybridization signals were detected within control samples. This study provides additional support for a pathogenic association of EcPV2 in oral SCC in horses.

Papillomaviruses (PVs) cause disease in humans, dogs, cats, and horses. While there are some differences, many aspects of the pathogenesis, presentation, and treatment of these diseases are similar between the four species. In this review, the PV-induced diseases of humans are compared to the similar diseases that develop in the companion animal species. By comparing with the human diseases, it is possible to make assumptions about some of the less common and less well-studied diseases in the veterinary species. In the first part of this review, the PV lifecycle is discussed along with the classification of PVs and the immune response to PV infection. The hyperplastic diseases caused by PVs are then discussed; including PV-induced cutaneous, anogenital, and oral warts within the four species.

Papillomaviruses (PVs) are well recognized to cause pre-neoplastic and neoplastic diseases in humans. Similarly, there is increasing evidence that PVs play a significant role in the development of pre-neoplastic and neoplastic diseases of the haired skin of dogs and cats, and the mucosa of horses. As the mechanisms by which PVs cause neoplasia are well studied in humans, it is valuable to compare the PV-induced neoplasms of humans with similar PV-associated neoplasms in the companion animal species. In the second part of this comparative review, the pre-neoplastic and neoplastic diseases thought to be caused by PVs in humans, dogs, cats, and horses are described. This includes PV-induced cutaneous plaques, cutaneous squamous cell carcinomas (SCCs) and mucosal SCCs within the four species. The review concludes with a discussion about the potential use of vaccines to prevent PV-induced diseases of dogs, cats, and horses.

Urogenital carcinoma in California sea lions (Zalophus californianus) is the most common cancer of marine mammals. Primary tumors occur in the cervix, vagina, penis, or prepuce and aggressively metastasize resulting in death. This cancer has been strongly associated with a sexually transmitted herpesvirus, otarine herpesvirus 1 (OtHV1), but the virus has been detected in genital tracts of sea lions without cancer and a causative link has not been established. To determine if OtHV1 has a role in causing urogenital carcinoma we sequenced the viral genome, quantified viral load from cervical tissue from sea lions with (n = 95) and without (n = 163) urogenital carcinoma, and measured viral mRNA expression using in situ mRNA hybridization (Basescope®) to quantify and identify the location of OtHV1 mRNA expression. Of the 95 sea lions diagnosed with urogenital carcinoma, 100% were qPCR positive for OtHV1, and 36% of the sea lions with a normal cervix were positive for the virus. The non-cancer OtHV1 positive cases had significantly lower viral loads in their cervix compared to the cervices from sea lions with urogenital carcinoma. The OtHV1 genome had several genes similar to the known oncogenes, and RNA in situ hybridization demonstrated high OtHV1 mRNA expression within the carcinoma lesions but not in normal cervical epithelium. The high viral loads, high mRNA expression of OtHV1 in the cervical tumors, and the presence of suspected OtHV1 oncogenes support the hypothesis that OtHV1 plays a significant role in the development of sea lion urogenital carcinoma.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5(+))-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9(+)Ki67(+))] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation. NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

Cutaneous papillomaviruses are oncogenic viruses that cause severe, persistent infections that can develop into skin cancers within ultraviolet (UV)-exposed skin of immunodeficient individuals, such as those with X-linked severe combined immunodeficiency (XSCID). A canine research model of XSCID exhibits a similar phenotype; these dogs develop severe canine papillomavirus 2 (CPV2) infections that often progress to cancer. Thus, the dog is a natural, spontaneous model to investigate cutaneous papillomavirus infections in immunodeficient patients. The human papillomavirus oncogene E6 contributes to cancer development, in part, by initiating degradation of the tumor suppressor protein p53, or by inhibiting upregulation of p53-dependent genes required within the cell growth arrest and apoptotic pathways, thereby leading to an accumulation of DNA damage required for oncogenesis. Currently, little is known about CPV2, and how it promotes cancer development. The aim of this study was to determine if CPV2 oncogene E6 similarly affects p53 upon activation by UV radiation, as well as the downstream p53-regulated genes necessary to control growth arrest and apoptosis. We determined that cutaneous CPV2 E6 does not degrade p53, or interfere with the upregulation of p53-regulated genes p21, Bax, Bak, or lncRNA-p21, suggesting that CPV2 may use a p53-independent mechanism to contribute to oncogenesis.

Objective To evaluate the ability of a bipolar sealing device (BSD) to seal canine bladder tissue and to determine the influence of suture augmentation on resistance to leakage of sealed partial cystectomies. Study design Ex vivo, simple randomized study. Sample population Urinary bladders harvested from canine cadavers (n = 23). Methods Partial cystectomy of the cranial third of each bladder was performed with a BSD. This seal was augmented with a simple continuous pattern of 4-0 polydioxanone in half of the specimens. A pressure transducer inserted through the ureter measured intraluminal pressure at initial leakage and catastrophic failure as dyed saline was infused via a catheter inserted through the urethra. Initial leakage pressure and pressure at catastrophic failure were compared between sutured and nonsutured sealed partial cystectomies. Results Sutured sealed cystectomies showed initial leakage at lower pressures compared to non-sutured cystectomies (8.6 vs. 17.7 mm Hg; P = .0365) but were able to sustain greater pressures at catastrophic failure (34.3 vs. 21.8 mm Hg; P = .007). Catastrophic failure occurred along the seam of all nonsutured sealed cystectomies and at the suture holes in 10 of the 12 sutured bladders. Conclusion Partial cystectomies were effectively sealed with a BSD in this canine cadaveric bladder model. Augmentation with a simple continuous suture pattern increased the pressure at which catastrophic leakage occurred but lowered initial leak pressure. Clinical significance This study provides evidence supporting the evaluation of BSD use for partial cystectomy in live animals. (c) 2020 The American College of Veterinary Surgeons

A red-eared slider with a chronic non-healing ulcerative shell lesion was diagnosed with cutaneous squamous cell carcinoma (SCC). The animal underwent surgical debulking and adjuvant hypofractionated radiation therapy. The lesion initially responded, with near-complete tumour regression, but then began growing again just a few months after finishing radiotherapy. Then, after several months with no additional tumour-directed therapy, the lesion again regressed. Five years post-irradiation and with no further treatment, the turtle now remains tumour-free. This unusual pattern of disease regression, followed by transient growth and then long-term local tumour control, suggests either a spontaneous remission or a pseudoprogression-like phenomenon. Careful clinical follow-up and reporting of future cases will aid in determining whether this pseudoprogression-like event was random, versus being a common component of the chelonian response to irradiation of cutaneous SCC.

Cutaneous papillomaviruses can cause severe, persistent infections and skin cancer in immunodeficient patients, including people with X-linked severe combined immunodeficiency (XSCID). A similar phenotype is observed in a canine model of XSCID; these dogs acquire severe cutaneous papillomavirus infections that can progress to cancer in association with canine papillomavirus type 2 (CPV2). This canine model system provides a natural spontaneous animal model for investigation of papillomavirus infections in immunodeficient patients. Currently, it is unknown if CPV2 can subvert the innate immune system and interfere with its ability to express antiviral cytokines, which are critical in the host defense against viral pathogens. The aim of the current study was to determine if the oncogenes E6 and E7 from CPV2 interfere with expression of antiviral cytokines in keratinocytes, the target cells of papillomavirus infections. We determined that E6 but not E7 interferes with the constitutive expression of some antiviral cytokines, including interferon (IFN)-β and the IFN-stimulated gene IFIT1. Both E6 and E7 interfere with the transcriptional upregulation of the antiviral cytokines in response to stimulation with the dsDNA Poly(dA:dT). In contrast, while E6 also interferes with the transcriptional upregulation of antiviral cytokines in response to stimulation with the dsRNA Poly(I:C), E7 interferes with only a subset of these antiviral cytokines. Finally, we demonstrated that E7 but not E6 abrogates signaling through the type I IFN receptor. Taken together, CPV2 E6 and E7 both impact expression of antiviral cytokines in canine keratinocytes, albeit likely through different mechanisms.

Squamous cell carcinoma (SCC) is the most common neoplasm of the equine stomach. However, the mechanisms underlying malignant transformation are unknown. As Equus caballus papillomavirus–2 (EcPV-2) is a likely cause of some genital SCCs, we hypothesized that EcPV-2 is associated with a subset of equine gastric SCCs. To this aim, we performed polymerase chain reaction (PCR) and in situ hybridization (ISH) for EcPV-2 E6/ E7 oncogenes on 11 gastric SCCs and on gastric samples from 15 control horses with no SCC. PCR for EcPV-2 was positive in 7/11 (64%) gastric SCCs; non-SCC gastric samples were all negative. Intense hybridization signals for EcPV-2 E6/E7 nucleic acid were detected by ISH within tumor cells in 5/11 (45%) gastric SCCs, including distant metastases. No hybridization signals were detected within any of the non-SCC gastric cases. This study provides support for a potential association between EcPV-2 infection and a subset of equine gastric SCC.

Papillomaviruses infect humans and animals, most often causing benign proliferations on skin or mucosal surfaces. Rarely, these infections persist and progress to cancer. In humans, this transformation most often occurs with high-risk papillomaviruses, where viral integration is a critical event in carcinogenesis. The first aim of this study was to sequence the viral genome of canine papillomavirus (CPV) 16 from a pigmented viral plaque that progressed to metastatic squamous cell carcinoma in a dog. The second aim was to characterize multiple viral genomic deletions and translocations as well as host integration sites. The full viral genome was identified using a combination of PCR and high throughput sequencing. CPV16 is most closely related to chipapillomaviruses CPV4, CPV9, and CPV12 and we propose CPV16 be classified as a chipapillomavirus. Assembly of the full viral genome enabled identification of deletion of portions of the E1 and E2/E4 genes and two viral translocations within the squamous cell carcinoma. Genome walking was performed which identified four sites of viral integration into the host genome. This is the first description of integration of a canine papillomavirus into the host genome, raising the possibility that CPV16 may be a potential canine high-risk papillomavirus type.

Background Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. Methods Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. Results Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. Conclusions Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.

Papillomaviruses (PVs) are found in many species and infect epithelial cells at both mucosal and cutaneous sites. PVs are generally species-specific and cause benign epithelial proliferations, often forming papillomas or plaques. Rarely, these infections can persist, allowing progression to in situ and invasive cancers. We describe herein a case of multiple cutaneous pigmented plaques from a California sea lion (Zalophus californianus) that progressed to in situ and invasive squamous cell carcinoma (SCC). The lesions were characterized by epithelial hyperplasia, hyperkeratosis, and hypergranulosis that bordered more dysplastic areas, and, at one site, bordered an invasive SCC. Immunohistochemistry for papillomavirus antigen revealed strong nuclear immunoreactivity within keratinocytes in the hyperplastic epidermis. PCR was performed using degenerate and specific primers to detect papillomavirus DNA. Specific primers were used to amplify Zalophus californianus papillomavirus 1 (ZcPV-1), the only sea lion papillomavirus known to date. We detected ZcPV-1 DNA within the pigmented plaque, and in both in situ and invasive SCC samples.