Chiara Urraro

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.

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.

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.

Productive Infection of Bovine Papillomavirus Type 2 in the Urothelial Cells of Naturally Occurring Urinary Bladder Tumors in Cattle and Water Buffaloes

Background Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. Methods and Findings E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium. Conclusion This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

Association of bovine papillomavirus type 2 (BPV-2) and urinary bladder tumours in cattle from the Azores archipelago.

Urinary bladder tumours in cattle are caused by chronic ingestion of bracken fern and BPV-1/2 infection. The objective of the present study was to assess if BPV-2 was present in urinary bladder lesions from cattle with chronic enzootic haematuria (CEH) from the Azores archipelago (Portugal), in order to gain further information regarding the epidemiologic distribution of this virus. Samples were analysed using PCR specific primers for BPV-2 DNA and an immunohistochemistry for BPV E5 oncoprotein detection. We found a 28% incidence rate of BPV-2 DNA in different types of tumours and cystitis cases (13 out of 46 samples). Tested positive samples for PCR were also positive for the viral E5 oncoprotein; protein immunolabeling was mainly detected within the cytoplasm of urothelial cells, displaying a juxtanuclear distribution. This is the first report of BPV-2 detection in urinary bladder tumours associated with CEH in cattle from the Azores archipelago.

Phosphatidylinositol-3-Kinase-AKT Pathway, Phospho-JUN and Phospho-JNK Expression in Spontaneously Arising Bovine Urinary Bladder Tumours

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). E5, the major BPV-1/2 oncoprotein, binds to the activated platelet-derived growth factor beta receptor (pPDGF-betaR), inducing cell transformation in vitro and spontaneously arising urinary bladder tumours. The aim of this study was to assess whether the 85 kDa regulatory subunit (p85) of the phosphatidylinositol-3-kinase (PI3K)-AKT pathway and other transforming signals phospho-JUN (pJUN) and phospho-JUN N-terminal kinases (pJNK) may be important in the development of BPV-associated urothelial carcinomas. A physical interaction between the pPDGF-betaR and PI3K was shown in four tumours and two samples of normal bladder tissue by co-immunoprecipitation and western blotting. There was greater expression of the PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of pPDGF-betaR in neoplastic compared with normal tissue. pJNK and pJUN were overexpressed in samples of tumour compared with normal mucosal tissue. These findings provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine urothelial carcinogenesis and contribute to understanding of the role of E5 oncoprotein in naturally occurring tumorigenesis.

Ferritin heavy chain (FHC) is up-regulated in papillomavirus-associated urothelial tumours of the urinary bladder in cattle.

The up-regulation of ferritin heavy chain (FHC) is reported in six papillary and in four invasive urothelial tumours of the urinary bladder of cattle grazing on mountain pastures rich in bracken fern. All tumours contained sequence of bovine papillomavirus type-2 (BPV-2) as determined by polymerase chain reaction (PCR) analyses and validated by direct sequencing of the amplified products. The oncoprotein E5 was also detected in these tumours by immunoprecipitation and by immunofluorescence and laser scanning confocal microscopy. Expression of FHC was evaluated by western blot analysis, reverse transcriptase (RT) PCR, real-time RT-PCR and immunohistochemistry. The oligonucleotide sequence of the bovine ferritin amplicons was identical to that of human ferritin. Nuclear overexpression of p65, an important component of nuclear factor kappaB (NF-kappaB) transcription factors, was also observed. These findings suggest that FHC up-regulation may be mediated by activation of NF-kappaB and that in turn this may be related to the resistance of bovine papillomavirus type-2 (BPV-2) infected urothelial cells to apoptosis.

Sigma-2 receptor expression in bovine papillomavirus-associated urinary bladder tumours.

The expression of sigma-2 receptors was investigated in nine urothelial tumours of the urinary bladder of cattle. Each tumour was associated with the presence of DNA of bovine papillomavirus type-2 (BPV-2) and expression of the E5 viral oncoprotein. Five tumours were classified as low-grade carcinoma on the basis of morphological criteria and calculation of mean nuclear area (MNA) and mean nuclear perimeter (MNP). Four tumours were classified as high-grade carcinoma. Sigma-2 receptors were overexpressed in both types of carcinoma. In control normal bovine bladder tissue the density of receptors (expressed as the B(max)) was 0.37 pmol/mg of protein. Low-grade carcinomas had a mean B(max) of 1.37+/-0.32 pmol/mg of protein (range 1.03-1.86) and in high-grade carcinomas the mean B(max) was 10.9+/-2.8 pmol/mg of protein (range 8.2-14). The difference in B(max) between low- and high-grade carcinomas was statistically significant (P=0.0001).

Bovine Papillomavirus Type 2 Infection and Microscopic Patterns of Urothelial Tumors of the Urinary Bladder in Water Buffaloes

Microscopic patterns of thirty-four urothelial tumors of the urinary bladder of water buffaloes from the Marmara and Black Sea Regions of Turkey are here described. All the animals grazed on lands rich in bracken fern. Histological diagnosis was assessed using morphological parameters recently suggested for the urinary bladder tumors of cattle. Papillary carcinoma was the most common neoplastic lesion (22/34) observed in this study, and low-grade carcinoma was more common (seventeen cases) than high-grade carcinoma (five cases). Papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and invasive carcinomas were less frequently seen. Carcinoma in situ (CIS) was often detected associated with some papillary and invasive carcinomas. De novo (primary) CIS was rare representing 3% of tumors of this series. A peculiar feature of the most urothelial tumors was the presence in the tumor stroma of immune cells anatomically organized in tertiary lymphoid organs (TLOs). Bovine papillomavirus type-2 (PV-2) E5 oncoprotein was detected by molecular and immunohistochemistry procedures. Early protein, E2, and late protein, L1, were also detected by immunohistochemical studies. Morphological and molecular findings show that BPV-2 infection contributes to the development of urothelial bladder carcinogenesis also in water buffaloes.

Lymphoepithelioma-like Carcinoma of the Urinary Bladder in a Cow Associated with Bovine Papillomavirus Type-2

Lymphoepithelioma-like carcinoma (LELCA) of the urinary bladder is reported in a 7-year-old cow that had grazed pasture rich in bracken fern and had suffered from severe intermittent haematuria from 3 to 4 years of age. On necropsy examination there were multiple haemorrhagic foci scattered over the mucosal surface of the urinary bladder. Microscopically there were nests, cords and sheets of neoplastic cells infiltrating the lamina propria and muscularis propria. These had a syncytial appearance with ill-defined cytoplasmic borders, large nuclei and prominent nucleoli. There was a prominent associated inflammatory infiltrate comprising lymphocytes and plasma cells with sparse histiocytes and granulocytes. Immunohistochemically, LELCA cells expressed cytokeratin but not vimentin. The LELCA was focally admixed with a concomitant papillary high-grade carcinoma that also infiltrated the lamina propria. A diffuse carcinoma in situ was also present. Bovine papillomavirus type-2 (BPV-2) DNA was amplified from frozen neoplastic tissue and from selected areas of formalin-fixed, paraffin wax-embedded tissue obtained by laser capture microdissection. Microbiological culture of a urine sample resulted in isolation of Weeksella virosa, Rhizobium radiobacter and Staphylococcus warneri. Flow cytometric analysis performed on blood mononuclear cells revealed down-regulation of a panel of markers including CD3, CD4, CD8alpha, CD45, MHC class I and MHC class II (HLA-DRalpha, HLA-DQ, HLA-DP). This report extends the spectrum of neoplastic urothelial lesions described in cattle and provides further evidence that some features of these tumours are similar to human counterparts.