Giuseppe Borzacchiello

Bovine papillomaviruses, papillomas and cancer in cattle.

Bovine papillomaviruses (BPV) are DNA oncogenic viruses inducing hyperplastic benign lesions of both cutaneous and mucosal epithelia in cattle. Ten (BPV 1-10) different viral genotypes have been characterised so far. BPV 1-10 are all strictly species-specific but BPV 1/2 may also infect equids inducing fibroblastic tumours. These benign lesions generally regress but may also occasionally persist, leading to a high risk of evolving into cancer, particularly in the presence of environmental carcinogenic co-factors. Among these, bracken fern is the most extensively studied. The synergism between immunosuppressants and carcinogenic principles from bracken fern and the virus has been experimentally demonstrated for both urinary bladder and alimentary canal cancer in cows whose diets were based on this plant. BPV associated tumours have veterinary and agricultural relevance in their own right, although they have also been studied as a relevant model of Human papillomavirus (HPV). Recent insights into BPV biology have paved the way to new fields of speculation on the role of these viruses in neoplastic transformation of cells other than epithelial ones. This review will briefly summarise BPV genome organization, will describe in greater detail the functions of viral oncoproteins, the interaction between the virus and co-carcinogens in tumour development; relevant aspects of immunity and vaccines will also be discussed.

Papillomavirus E5: the smallest oncoprotein with many functions

Papillomaviruses (PVs) are established agents of human and animal cancers. They infect cutaneous and mucous epithelia. High Risk (HR) Human PVs (HPVs) are consistently associated with cancer of the uterine cervix, but are also involved in the etiopathogenesis of other cancer types. The early oncoproteins of PVs: E5, E6 and E7 are known to contribute to tumour progression. While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems. The Bovine PV (BPV) system has been the most useful animal model in understanding the oncogenic potential of PVs due to the pivotal role of its E5 oncoprotein in cell transformation. This review will highlight the differences between HPV-16 E5 (16E5) and E5 from other PVs, primarily from BPV. It will discuss the targeting of E5 as a possible therapeutic agent.

Detection of bovine papillomavirus type 2 in the peripheral blood of cattle with urinary bladder tumours : possible biological role

Bovine papillomavirus type 2 (BPV-2) infection has been associated with urinary bladder tumours in adult cattle grazing on bracken fern-infested land. In this study, we investigated the simultaneous presence of BPV-2 in whole blood and urinary bladder tumours of adult cattle in an attempt to better understand the biological role of circulating BPV-2. Peripheral blood samples were collected from 78 cattle clinically suffering from a severe chronic enzootic haematuria. Circulating BPV-2 DNA was detected in 61 of them and in two blood samples from healthy cows. Fifty of the affected animals were slaughtered at public slaughterhouses and neoplastic proliferations in the urinary bladder were detected in all of them. BPV-2 DNA was amplified and sequenced in 78 % of urinary bladder tumour samples and in 38.9 % of normal samples as a control. Circulating episomal BPV-2 DNA was detected in 78.2 % of the blood samples. Simultaneous presence of BPV-2 DNA in neoplastic bladder and blood samples was detected in 37 animals. Specific viral E5 mRNA and E5 oncoprotein were also detected in blood by RT-PCR and Western blot/immunocytochemistry, respectively. It is likely that BPV-2 can persist and be maintained in an active status in the bloodstream, in particular in the lymphocytes, as a reservoir of viral infection that, in the presence of co-carcinogens, may cause the development of urinary bladder tumours.

Bovine papillomavirus E5 oncoprotein binds to the activated form of the platelet-derived growth factor beta receptor in naturally occurring bovine urinary bladder tumours.

Studies regarding the functions of the bovine papillomavirus (BPV) E5 oncoprotein in vivo are lacking and no E5-mediated mechanism underlying epithelial carcinogenesis is known. We have shown that BPV-2 DNA is present in the majority of naturally occurring urinary bladder tumours of cattle and that E5 is expressed in the cancer cells. Here we show that the interaction between the platelet-derived growth factor (PDGF) β receptor and BPV E5, described in vitro in cultured cells, takes place in vivo in bovine urinary bladder cancers. In these cancers, E5 and PDGF β receptor colocalize, as shown by confocal microscopy, and physically interact, as shown by coimmunoprecipitation. Furthermore, the PDGF β receptor associated with E5 is highly phosphorylated, suggesting the functional activation of the receptor upon E5 interaction. Our results demonstrate, for the first time, that E5–PDGF β receptor interaction occurs during the natural history of bovine urinary bladder tumours, suggesting an important role for E5 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E5. Successful bladder tumour regression would provide a valuable model for therapeutic vaccination against papillomavirus-associated tumours.

PBMCs are additional sites of productive infection of bovine papillomavirus type 2.

Bovine papillomavirus type 2 (BPV-2) is an oncogenic virus infecting both epithelial and mesenchymal cells. Its life cycle, similar to other papillomaviruses (PVs), appears to be linked to epithelial differentiation. Human and bovine PVs have been known to reside in a latent, episomal form in PBMCs; therefore, it is believed that blood cells, like all mesenchymal cells, function as non-permissive carriers. Here, for the first time in veterinary and comparative medicine, the BPV-2 E5 oncoprotein and the major structural L1 capsid protein, known to be expressed only in productive infections, were shown to occur in defined subsets of PBMCs. E5 oncoprotein was detected in sorted T- and B-cells as well as in monocytes by flow cytometry and Western blot analysis. However, CD4(+) and CD8(+) lymphocytes appeared to be the main circulating targets of the virus, thus possibly representing the most important reservoir of active BPV-2 in blood. L1 protein was identified by flow cytometry in a population of blood cells recognized as lymphocytes by morphological scatter properties. Western blot analysis was performed on lysates obtained from the sorted subpopulations of PBMCs and detected L1 protein in CD4(+) and CD8(+) cells only. Thus, this study showed that CD4(+) and CD8(+) lymphocytes are permissive for BPV-2 and are new, hitherto unknown sites of productive PV infection. In light of these observations, the life cycle of PVs needs to be revisited to gain novel insights into the epidemiology of BPV infection and the pathogenesis of related diseases.

Expression of Platelet-derived Growth Factor-β Receptor and Bovine Papillomavirus E5 and E7 Oncoproteins in Equine Sarcoid

Equine sarcoids are benign fibroblastic skin tumours that are recognized throughout the world. Infection with bovine papillomavirus (BPV) types 1 and 2 has been implicated as a major factor in disease development; however, the cellular mechanisms underlying fibroblast transformation remain poorly defined. The present study further characterizes aspects of the association with BPV in 15 equine sarcoids. BPV DNA was demonstrated in 12/15 tumours collected from different areas of Italy. Nine of these 12 tumours expressed the BPV oncoproteins E5 and E7, but these oncoproteins were not expressed by normal equine cells. The BPV E5 protein is known to bind to the platelet-derived growth factor-beta receptor (PDGF-betaR) and this molecule was expressed by 11 of the 12 sarcoids in which E5 was demonstrated. These findings add further weight to the theory that BPV and the PDGF-betaR may have a role in the pathogenesis of this disease.

Productive Infection of Bovine Papillomavirus Type 2 in the Placenta of Pregnant Cows Affected with Urinary Bladder Tumors

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Inoculation of young horses with bovine papillomavirus type 1 virions leads to early infection of PBMCs prior to pseudo-sarcoid formation

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11-32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

Calpain3 is expressed in a proteolitically active form in papillomavirus-associated urothelial tumors of the urinary bladder in cattle.

Background Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle. Methods and Findings Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca2+-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting. Conclusion The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

Bovine papillomavirus type 2 infects the urinary bladder of water buffalo (Bubalus bubalis) and plays a crucial role in bubaline urothelial carcinogenesis

Bovine papillomavirus type 2 (BPV-2) has been shown to infect and play a role in urinary bladder carcinogenesis of buffaloes grazed on pastures with ferns from the Marmara and Black Sea Regions of Turkey. BPV-2 DNA has been found in both neoplastic and non-neoplastic lesions of the urinary bladder. Furthermore, this virus may be a normal inhabitant of the urinary bladder since BPV-2 DNA has also been detected in clinically normal buffaloes. The viral activation by fern immunosuppressant or carcinogen may trigger the urothelial cell transformation. The E5 oncoprotein was solely detected in urothelial tumours and appeared to be co-localized with the overexpressed and phosphorylated platelet derived growth factor (PDGF) β receptor in a double-colour immunofluorescence assay. Our results indicate that the E5-PDGF β receptor interaction also occurs in spontaneous tumours of the bubaline urinary bladder, revealing an additional role of BPV-2 in bladder carcinogenesis of buffaloes.