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Short Communication |
1 Equine Centre, Veterinary University Vienna, Vienna, Austria
2 Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Berne, Berne, Switzerland
3 Ontario Veterinary College, University of Guelph, ON, Canada
4 Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University Vienna, Vienna, Austria
Correspondence
Sabine Brandt
sabine.brandt{at}vu-wien.ac.at
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10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from enciphered PBMC DNA derived from 66 horses, revealing E5 in the PBMCs of three individuals with confirmed sarcoids, whereas the remaining 63 sarcoid-free animals were negative for this gene. L1 could not be detected in any PBMC DNA, suggesting either deletion or interruption of this gene in PBMCs of BPV-1/-2-infected equines. These results support the hypothesis that PBMCs may serve as host cells for BPV-1/-2 DNA and contribute to virus latency. |
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). In contrast to BPV-induced bovine papillomas, which usually resolve spontaneously, equine sarcoids mostly persist and tend to recrudesce following treatment. In general, sarcoids do not metastasize, yet their localization and progression to more aggressive types can compromise the use and welfare of affected animals (Scott & Miller, 2003).
An aetiological association of BPV with sarcoids was first suspected when inoculation with bovine wart extract produced transient sarcoid-like lesions in horses (Olson & Cook, 1951; Voss, 1969). BPV DNA was first demonstrated in sarcoids by Southern blotting (Lancaster et al., 1979; Amtmann et al., 1980; Trenfield et al., 1985). Using the more sensitive PCR, BPV DNA has been detected in up to 100 % of investigated sarcoids (Otten et al., 1993; Bloch et al., 1994; Carr et al., 2001a, b; Martens et al., 2001a, b; Bogaert et al., 2005, 2008) and also in apparently intact skin of sarcoid-bearing individuals, leading to the speculation that BPV might reside latently in fibroblasts until factors such as trauma initiate its transcriptional and hence transforming activity (Carr et al., 2001a). There is evidence that BPV DNA cannot be found in sarcoid-free horses or non-sarcoid equine tumours (Otten et al., 1993; Carr et al., 2001a, b). However, the presence of viral DNA in skin swabs of unaffected horses has been reported recently (Bogaert et al., 2005, 2008). In addition, BPV-1 DNA (Angelos et al., 1991; Chambers et al., 2003a) and early gene transcripts have been demonstrated in some cases of dermatitis (Yuan et al., 2007).
Although viral genes are intralesionally transcribed (Nasir & Reid, 1999; Carr et al., 2001b; Nixon et al., 2005; Bogaert et al., 2007), intact virions have not been detected in sarcoids so far. Hence, the disease is understood as the result of an abortive infection where BPV exists episomally (Amtmann et al., 1980; Lancaster, 1981).
The mode of BPV transmission within and between animals is still unclear. Virus may be propagated by direct contact or via contaminated habitual surroundings such as tack, barns or stable walls (Chambers et al., 2003b; Bogaert et al., 2005). The predilection for sarcoid development at wound sites also suggests that insects infesting sites of trauma may act as vectors. Indeed, viral DNA has been detected in face flies feeding on sarcoids (Kemp-Symonds, 2000). Blood might be another reservoir of viral DNA contributing to the propagation of the disease. BPV DNA has been demonstrated in whole blood of infected cattle (Campo et al., 1994; Campo, 1998; De Freitas et al., 2003; Wosiacki et al., 2005), suggesting vertical virus transmission via the blood stream. Following blood transfusion from papilloma-bearing to virus-free cattle, BPV DNA was detected in peripheral blood mononuclear cells (PBMCs) of recipient cows and their progeny, supporting the concept that BPV can be transmitted in utero (Stocco dos Santos et al., 1998). Human papillomavirus (HPV) DNA has been detected in PBMCs (Pao et al., 1991; Bodaghi et al., 2005), serum (Liu et al., 2001) and plasma (Dong et al., 2002) of human patients affected by HPV-associated cancers. This finding was initially interpreted as originating from disseminated tumour metastases. However, HPV DNA has also been detected in PBMCs of clinically unremarkable children that had acquired a human immunodeficiency virus (HIV) infection via blood transfusion or vertical transmission, thus suggesting that HPV had been co-transmitted by PBMCs (Bodaghi et al., 2005).
BPV DNA has not been demonstrated in the blood of BPV-infected equids so far (Angelos et al., 1991; Nasir et al., 1997; Bogaert et al., 2008). Based on the hypothesis that PBMC-derived BPV DNA may have escaped detection due to extremely reduced virus loads, we reassessed this issue by establishing a highly sensitive PCR protocol for detection of the BPV-1/-2 genes E5 and L1. Cloned full-length BPV-1 DNA (plasmid BPV1-pML) with an initial concentration of 108 ng µl–1, equivalent to 1010 molecules µl–1, was serially diluted 10-fold (109–101 genome copies) in virus-free genomic DNA (170 ng µl–1) obtained from an 
3 mm3 skin biopsy of a healthy horse using a DNeasy Tissue Extraction kit (Qiagen). Subsequently, 1 µl sample aliquots were subjected to E5 and L1 PCR using BPV-1/-2 consensus primers selected from published BPV-1 (GenBank accession no. X02346
[GenBank]
) and BPV-2 (GenBank accession no. M20219
[GenBank]
) sequences. E5-specific primers (5'B1/2-E5: 5'-CACTACCTCCTGGAATGAACATTTCC-3'; 3'B1/2-E5: 5'-CTACCTTWGGTATCACATCTGGTGG-3') were designed for amplification of a 499 (BPV-1) or 497 bp (BPV-2) fragment spanning the E5 open reading frame (ORF). L1-specific primers (5'B1/2-L1: 5'-GCTAAGCAACAACAGATTCTGTTGC-3'; 3'B1/2-L1: 5'-TCAGCCATTTTGAGGTAGTCTGG-3') were designed for amplification of a 266 bp region of the major capsid gene L1. PCR was carried out in 0.5 ml µlTI Ultra PCR tubes (Sorenson Bioscience), each containing 9.5 % DMSO, 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.5 mM each dNTP, 100 pmol sense and antisense primer, and 1 µl DNA template in a volume of 49 µl, using two drops of mineral oil as a top layer. Reaction tubes were placed unlocked in an Eppendorf Mastercycler. Following a manual hot start at 95 °C for 5 min and the addition of 1 U Taq polymerase (Roche Life Science), tubes were closed and an amplification program consisting of seven touch-down cycles [92 °C for 30 s/65–56 °C for 45 s (–1.5 °C per cycle)/72 °C for 45 s], followed by 40 standard cycles (92 °C for 30 s/56 °C for 45 s/72 °C for 45 s) was performed. PCR products were visualized on 2 % Tris/acetate agarose gels by ethidium bromide staining. As shown in Fig. 1(a), both reactions were positive for all plasmid dilutions (109–101 copies), thus revealing a PCR detection limit of 10 copies for E5 and L1. A plasmid-free negative control reaction (dilution 0) was included in each experiment.
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; Table 1). Sarcoid DNA of an individual with a determined BPV-1-infection (Jsa) was used successfully as positive control. To confirm specificity, one PBMC-derived amplicon (Fig. 1b, lane 2) was cloned and sequenced, revealing four silent point mutations compared with BPV-1 E5 laboratory clones with 100 % identity to the predicted BPV-1 E5 protein sequence Swiss III (GenBank accession no. AY232259
[GenBank]
; Chambers et al., 2003a).
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; Table 1).
Finally, blind PCRs for E5 and L1 were carried out from enciphered PBMC DNA isolates (1–66) obtained from 66 co-stabled horses (donated by Monika Seltenhammer, our laboratory). The success of DNA purification was confirmed by β-actin PCR (not shown) and photometry, the latter revealing a mean DNA concentration of 320 ng µl–1. E5 PCR from 2 µl DNA aliquots was positive for 3/66 DNA specimens (PBMC DNA isolates 20, 24 and 33), as well as for sarcoid- (Jsa) and PBMC-derived (Bb, Db and Eb) positive controls. No amplification products were obtained for the negative (Fs and Hb) and no-template controls (Fig. 2; Table 1). The L1 PCR was negative for all tested DNA specimens except for the sarcoid DNA-positive control (not shown). Following decoding, clinical examination and analysis of the medical records provided revealed the exclusive presence of sarcoids in E5-positive individuals 20, 24 and 33, whereas no BPV-related malignancies could be determined for the remaining 63 individuals. Sample specifications and the PCR results are summarized in Table 1.
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Quantitative E5 PCR recently carried out by us has revealed <100 E5 molecules per 1.2x105 PBMCs. The occurrence of such small amounts of E5 in white blood cells (mean of 1 copy per 1000 cells) in combination with less sensitive detection or PBMC extraction methods may explain previous failure to demonstrate them.
The intralesional presence of BPV-1/-2 E5 DNA and transcripts has been well documented (Nasir & Reid, 1999; Carr et al., 2001b; Chambers et al., 2003a; Bogaert et al., 2007), supporting an active role of E5 in sarcoid formation. We have detected E5 DNA in the PBMCs of BPV-1/-2-affected horses, suggesting an as yet unknown concurrence with morbidity. E5 was also identified in a histologically diagnosed periocular SCC. A novel equine papillomavirus termed EcPV-2 was recently identified in genital (Scase, 2007) but not in ocular SCC, suggesting an association of as yet unidentified papillomaviruses with ocular SCC. BPV-1/-2 E5 DNA was detected equally in PBMCs and perilesional tissue of a sarcoid-free mare affected by a vaccination abscess. It is not yet clear whether infection in this horse was accidental or contributed to abscess formation.
The L1 capsid gene-specific 266 bp sequence could not be detected in E5-containing PBMCs, the abscess or the periocular SCC, suggesting that L1 may be partially or totally deleted from the viral genome. Absence of the cottontail rabbit papillomavirus L1 ORF has been shown to compromise papilloma formation (Nasseri et al., 1989). However, transfection of murine cells with an L1-deleted BPV-1 genome resulted in tumorigenic transformation (Lowy et al., 1980). Moreover, in vivo transforming activity has been observed for naturally occurring L1 deletion mutants of BPV-1 (Angelos et al., 1991), HPV-6a, HPV-5 and HPV-8 (Ostrow et al., 1982, 1987; Deau et al., 1991; Suzuki et al., 1995), suggesting that L1 may not be required for episomal maintenance and transforming functions in abortive papillomavirus infection.
Alternatively, L1 might be interrupted or lost due to integration of viral DNA into the host cell genome. Integration of cancer-associated HPV types is assumed to correlate with tumour malignancy, possibly because integration occurs at oncogene sites or disrupts tumour suppressor genes (Popescu et al., 1990; Greenspan et al., 1997; Ferber et al., 2003a, b). However, viral integration has never been observed in BPV-infected ungulates (Lancaster, 1981). In an experimental model, BPV-1 transgenic mice developed fibropapillomas harbouring viral episomes, whereas normal tissue contained integrated BPV-1 DNA (Lacey et al., 1986), suggesting that the BPV-1 genome can undergo excision events correlating with tumour formation. By analogy, it is conceivable that BPV integrants may be maintained in PBMCs until excision occurs following interaction with as yet unknown factors.
The presence of BPV DNA in PBMCs suggests a possible contribution to virus spread. Given that whole infectious virus is assumed to produce de novo infection (Ragland & Spencer, 1969) and that horses are considered to be non-permissive hosts for BPV (Chambers et al., 2003b), it seems unlikely that infected PBMCs are involved in horizontal BPV transmission. However, virus may spread in utero from infected mares to their foals, as has been shown in cattle. Moreover, infected PBMCs may propagate disease within one individual, as they migrate to sites of inflammation where they may take up the virus and function as a carrier. Alternatively, PBMC infection may trigger disease by negatively affecting their immunological functions. Further studies are warranted to elucidate the biological and pathological significance of our findings.
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Received 6 November 2007;
accepted 19 February 2008.
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