Oral fibropapillomatosis and epidermal hyperplasia of the lip in newborn lambs associated with bovine Deltapapillomavirus.

Congenital fibropapillomatosis of the gingiva and oral mucosa and epidermal hyperplasia of the lip are described, for the first time, in two newborn lambs. Expression of the E5 oncoprotein of bovine deltapapillomavirus types 2 (BPV-2) and -13 (BPV-13) was detected in both fibropapillomas and the hyperplastic epidermal cells suggesting the BPV infection was the cause of the proliferative lesions. No DNA sequences of BPV-1 and BPV-14 were detected. Both BPV-2 and BPV-13 DNA were also amplified from peripheral blood mononuclear cells (PBMCs) of the newborn lambs' dams. The concordance between BPV genotypes detected in the blood of dam and the oral and skin pathological samples of their offspring suggests that a vertical hematogeneous transmission was most likely source of BPV infection. Immunoblotting revealed the presence of E5 dimers allowing the viral protein to be biologically active. E5 dimers bind and activate the platelet derived growth factor ß receptor (PDGFßR), a major molecular mechanism contributing to disease. The detection of E5 protein within the proliferating cells therefore adds further evidence that the BPV infection was the cause of the proliferative lesions seen in these lambs. This is the first evidence of vertical transmission of BPVs in sheep resulting in a clinical disease.

Nuclear myosin 1 associates with papillomavirus E2 regulatory protein and influences viral replication.

Nuclear myosin 1c (NM1) associates with RNA polymerases and is a partner in the chromatin remodeling complex B-WICH. This complex, which also contains WSTF and SNF2h proteins, is involved in transcriptional regulation. We report herein that papillomavirus protein E2 binds to NM1 and co-precipitates with the WSTF and SNF2h proteins. Our data suggest that E2 associates with the cellular B-WICH complex through binding to NM1. E2 and NM1 associate via their N-terminal domains and this interaction is ATP dependent. The cellular multifunctional protein Brd4 and beta-actin are also present in the NM1-E2 complex. NM1 downregulation by siRNA increases the replication of the BPV1 and HPV5 genomes but does not affect HPV18 genome replication. These results suggest that the B-WICH complex may play a role in the papillomavirus life cycle through NM1 and E2 protein interaction.

Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells.

The bovine papillomavirus E1 helicase is essential for viral replication. In dividing cells, DNA replication maintains, but does not increase, the viral genome copy number. Replication is limited by low E1 expression and an E1 nucleocytoplasmic shuttling mechanism. Shuttling is controlled in part by phosphorylation of E1 by cellular kinases. Here we investigate conserved sites for phosphorylation by kinase CK2 within the E1 nuclear localization signal. When these CK2 sites are mutated to either alanine or aspartic acid, no change in replication phenotype is observed, and there is no effect on the subcellular distribution of E1, which remains primarily nuclear. This demonstrates that phosphorylation of E1 by CK2 at these sites is not a factor in regulating viral DNA replication in dividing cells.

Detection and quantification of bovine papillomavirus type 2 in urinary bladders and lymph nodes in cases of Bovine Enzootic Hematuria from the endemic region of Azores.

Bovine Enzootic Hematuria (BEH) is a disease with a severe impact on production indexes and characterized by the development of bovine urinary bladder tumors, particularly in the Azores archipelago. The purpose of this study was to investigate and quantify BPV2 tissue distribution in bovine urinary bladder tumors, normal bladders, and iliac lymph nodes of cattle from the Azores. A real-time PCR system targeting the L1 gene was developed and allowed for the specific detection of the virus. BPV2 DNA was detected in a large proportion of the samples tested, both from neoplastic and healthy tissues, indicating that this virus is very prevalent in the bovine population of the Azores. Moreover, all types of tissues tested were positive, confirming a wide viral distribution within the infected animal. Bovine cutaneous papillomas sampled from Portuguese mainland dairy cattle were used as controls. Viral load ranged between 2.2×10(4) copies/cell in the skin papillomas, and 0.0002 copies/cell in the urinary bladders tumors from the Azores. This is the first report presenting quantitative data on BPV2 infection in bovine urinary bladder lesions from the Azores. This approach will provide a useful tool to evaluate the role of BPV2 not only in the pathogenesis BEH but also in cell transformation mechanisms.

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V1-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

BACKGROUND: Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. METHODS AND FINDINGS: In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor ß receptor. PDGFßR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V1-ATPase subunit D", a component of the central stalk of the V1-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. CONCLUSION: For the first time, a tri-component complex composed of E5/PDGFßR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V0-ATPase sector. We suggest that the E5/PDGFßR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor ß receptor into an inhibitor.

Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.

Expression of platelet derived growth factor ß receptor, its activation and downstream signals in bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are benign skin tumours formed by proliferation of epidermal keratinocytes and dermal fibroblasts caused by bovine papillomaviruses (BPVs). BPV E5 oncoprotein plays a key role in neoplastic cell transformation by specifically binding to the platelet derived growth factor beta receptor (PDGFßR) causing its phosphorylation and activation of proliferation and survival signal transduction pathways, among these phosphatidyl inositol-3-kinase (PI3K)/Akt and Ras-mitogen-activated-protein-kinase-Erk (Ras-MAPK-Erk) pathways. The aim of this study was to investigate the expression of PDGFßR, its phosphorylation status and expression of the downstream molecules phospho-Akt (pAkt) and phospho-Erk (pErk), in naturally occurring bovine cutaneous fibropapillomas. By immunohistochemistry on serial sections we showed cytoplasmic co-expression of the PDGFßR and E5 protein in neoplastic tissue. Western blot analysis revealed that PDGFßR was phosphorylated in higher amount in tumour samples compared to normal skin. pAkt, but not pErk, was also overexpressed in tumour samples. These findings may provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine fibropapillomas and contribute to understanding the molecular scenario underlying BPV induced tumourigenesis.

Characterisation of early and late bovine papillomavirus protein expression in equine sarcoids.

Sarcoids are common skin tumours of horses and donkeys that are characterised by persistent proliferation of dermal fibroblasts associated with the presence of bovine papillomavirus (BPV) DNA. Some early BPV proteins have been demonstrated within sarcoids and RNA containing both early and late transcripts is present, yet it remains unclear whether late replication of BPV, culminating in the production of infectious virus particles, can occur in equids. Here we report that BPV1 RNA isolated from equine sarcoids encodes a unique deletion of four residues within the L2 protein suggesting a novel variant of virus has evolved in equines. Such viral evolution would require the production and transmission of virus particles among horses with sarcoids. Quantitative RT-PCR demonstrated the presence of mRNA transcripts containing early gene message in sarcoid tissues and BPV-E2 early virus antigen was detected by immunofluorescence in the nuclei of dermal fibroblasts, but no E2 expression could be detected within the overlying epidermis where productive virus replication would be expected to occur. Although immunohistochemistry clearly detected late virus proteins in the nuclei of dermal cells from samples of bovine papillomas, no late protein expression was detected in formalin-fixed tissue from equine sarcoids; either in the dermis or epidermis. Moreover, quantitative RT-PCR demonstrated that late gene mRNA represented <0.3% of the transcribed BPV RNA. We conclude that BPV does not undergo productive infection in the epidermis overlying equine sarcoids at levels comparable with that occurring in its natural bovine host.

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.

Molecular and epigenetic analysis of the fragile histidine triad tumour suppressor gene in equine sarcoids.

BACKGROUND: Sarcoids are peculiar equine benign tumours. Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection. Little is known about the molecular interplay between viral infection and neoplastic transformation. The data regarding papillomavirus infections in human species show the inactivation of a number of tumour suppressor genes as basic mechanism of transformation. In this study the putative role of the tumour suppressor gene Fragile Histidine Triad (FHIT) in sarcoid tumour was investigated in different experimental models. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissue. RESULTS: Nine paraffin embedded sarcoids and sarcoid derived cell lines were analysed for the expression of FHIT protein by immunohistochemistry, immunofluorescence techniques and western blotting. These analyses revealed the absence of signal in seven out of nine sarcoids. The two sarcoid derived cell lines too showed a reduced signal of the protein. To investigate the causes of the altered protein expression, the samples were analysed for the DNA methylation profile of the CpG island associated with the FHIT promoter. The analysis of the 32 CpGs encompassing the region of interest showed no significative differential methylation profile between pathological tissues and cell lines and their normal counterparts. CONCLUSION: This study represent a further evidence of the role of a tumour suppressor gene in equine sarcoids and approaches the epigenetic regulation in this well known equine neoplasm. The data obtained in sarcoid tissues and sarcoid derived cell lines suggest that also in horse, as in humans, there is a possible involvement of the tumour suppressor FHIT gene in BPV induced tumours. DNA methylation seems not to be involved in the gene expression alteration. Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.

Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6.

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.

Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR.

The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor ß receptor through transmembrane (TM) helix-helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively 15N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in 15N-(1)H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (ΔGx°=-3.05 kcal mol(-1)) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.

Cell-type specific transcriptional activities among different papillomavirus long control regions and their regulation by E2.

This study systematically examined the viral long control region (LCR) activities and their responses to E2 for human papillomavirus (HPV) types 11, 16, and 18 as well as bovine papillomavirus 1 (BPV1) in a number of different cell types, including human cervical cancer cell lines, human oral keratinocytes, BJ fibroblasts, as well as CV1 cells. The study revealed cell- and virus-type specific differences among the individual LCRs and their regulation by E2. In addition, the integration of the LCR into the host genome was identified as a critical determinant for LCR activity and its response to E2. Collectively, these data indicate a more complex level of transcriptional regulation of the LCR by cellular and viral factors than previously appreciated, including a comparatively low LCR activity and poor E2 responsiveness for HPV16 in most human cells. This study should provide a valuable framework for future transcriptional studies in the papillomavirus field.

Bovine papillomavirus type 1 oncoprotein E5 inhibits equine MHC class I and interacts with equine MHC I heavy chain.

Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 cells were morphologically transformed, elongated with many pseudopodia and capable of forming foci. Equine major histocompatibility complex class I (MHC I) was inhibited in these cells at least at two levels: transcription of MHC I heavy chain was inhibited and the MHC I complex was retained in the Golgi apparatus and prevented from reaching the cell surface. We conclude that, as in bovine cells and tumours, E5 is a player in the transformation of equine cells and the induction of sarcoids, and a potential major cause of MHC I downregulation and hence poor immune clearance of tumour cells.

Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts.

Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targeting of E2 provides a powerful strategy to eliminate BPV-1 genomes and induce cell death in BPV-1 transformed cells.

Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas.

Papillomas and fibropapillomas may occur in the skin and in different organs in animals. Ten different genotypes of bovine papillomavirus (BPV) have been identified. BPV-1 through BPV-10 are all strictly species-specific, but BPV-1/2 may also infect other species such as equids, inducing fibroblastic tumors. BPV-1 and BPV-2 are associated with fibropapillomas in cattle; these tumors are formed by excessive proliferation of virus-infected dermal fibroblasts and epidermal keratinocytes. Nine water buffalo (Bubalus bubalis) were examined for the presence of multiple cutaneous and perivulvar tumors. Cutaneous and perivulvar fibropapillomatosis were confirmed histologically. Negative-stain transmission electron microscopic examination revealed papillomavirus-like particles in the fibropapillomas, and papillomaviral DNA was also detected by the polymerase chain reaction. The amplified long control region (LCR) DNA sequence was identical to that of BPV-1. The BPV-1 E5 oncoprotein was strongly expressed in the tumor cells thus confirming a causal role of the virus. This article represents the first report of cutaneous, perivulvar, and vulvar fibropapilloma associated with BPV-1 infection in the water buffalo and describes another example of cross-species infection by BPV-1.

Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo.

Papillomaviruses are small DNA viruses that induce epithelial lesions in their host. The viral life cycle is regulated by the family of proteins encoded by the E2 open reading frame. In addition to the full-length E2 protein, the BPV-1 genome encodes two truncated E2 proteins, E2C and E8/E2, which maintain the DNA-binding-dimerization domains, but lack the activation domain. Heterodimers formed between the full-length E2 and truncated E2 proteins serve as activators of E2-dependent transcription and papillomavirus DNA replication. We show that the single activation domain of E2 is sufficient for interaction with viral helicase E1 and for initiation of DNA replication from different papillomavirus origins. Single-chain E2 heterodimer is able to activate papillomavirus DNA replication in the context of entire BPV genome in the absence of other E2 proteins. These data suggest that E2 heterodimers with single activation domain are functional in initiation of papillomavirus replication in vivo.

The bovine papillomavirus E5 protein and the PDGF beta receptor: it takes two to tango.

The extremely hydrophobic, 44-amino acid bovine papillomavirus (BPV) E5 protein is the smallest known oncoprotein, which orchestrates cell transformation by causing ligand-independent activation of a cellular receptor tyrosine kinase, the platelet-derived growth factor beta receptor (PDGFbetaR). The E5 protein forms a dimer in transformed cells and is essentially an isolated membrane-spanning segment that binds directly to the transmembrane domain of the PDGFbetaR, inducing receptor dimerization, autophosphorylation, and sustained mitogenic signaling. There are few sequence constraints for activity as long as the overall hydrophobicity of the E5 protein and its ability to dimerize are preserved. Nevertheless, the E5 protein is highly specific for the PDGFbetaR and does not activate other cellular proteins. Genetic screens of thousands of small, artificial hydrophobic proteins with randomized transmembrane domains inserted into an E5 scaffold identified proteins with diverse transmembrane sequences that activate the PDGFbetaR, including some activators as small as 32-amino acids. Analysis of these novel proteins has provided new insight into the requirements for PDGFbetaR activation and specific transmembrane recognition in general. These results suggest that small, transmembrane proteins can be constructed and selected that specifically bind to other cellular or viral transmembrane target proteins. By using this approach, we have isolated a 44-amino acid artificial transmembrane protein that appears to activate the human erythropoietin receptor. Studies of the tiny, hydrophobic BPV E5 protein have not only revealed a novel mechanism of viral oncogenesis, but have also suggested that it may be possible to develop artificial small proteins that specifically modulate much larger target proteins by acting within cellular or viral membranes.

Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability.

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions.

A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein.

Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 perilesional/intact skin biopsies, and 1 serum. Tissue extracts from sarcoid-free equines served as controls. IC/PCR scored positive in 14/24 (58.3%) specimens obtained from sarcoid-patients, but negative for controls. Quantitative IC/PCR demonstrated <125 immunoprecipitable viral genomes/50 microl extract for the majority of specimens. Moreover, full-length BPV-1 genomes were detected in a complex with L1 proteins. These complexes may correspond to virion precursors or intact virions.