Download Peripheral blood mononuclear cells represent a reservoir of bovine

Journal of General Virology (2008), 89, 1390–1395
Short
Communication
DOI 10.1099/vir.0.83568-0
Peripheral blood mononuclear cells represent a
reservoir of bovine papillomavirus DNA in
sarcoid-affected equines
Sabine Brandt,1 Rhea Haralambus,1,2 Angelika Schoster,3
Reinhard Kirnbauer4 and Christian Stanek1
Correspondence
1
Sabine Brandt
2
[email protected]
Equine Centre, Veterinary University Vienna, Vienna, Austria
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
Received 6 November 2007
Accepted 19 February 2008
Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid
pathogenesis. However, the mode of virus transmission and the presence of latent infections are
largely unknown. This study established a PCR protocol allowing detection of ¡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.
Bovine papillomaviruses types 1 and 2 (BPV-1 and -2) are
chiefly involved in the pathogenesis of locally aggressive
cutaneous lesions termed sarcoids that frequently affect
horses, donkeys and mules (Olson & Cook, 1951). 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
1390
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
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BPV-1/-2 DNA in PBMCs of sarcoid-affected equines
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 BPVinfected 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 ml21, equivalent to 1010 molecules ml21, was
serially diluted 10-fold (109–101 genome copies) in virusfree genomic DNA (170 ng ml21) obtained from an
~3 mm3 skin biopsy of a healthy horse using a DNeasy
Tissue Extraction kit (Qiagen). Subsequently, 1 ml sample
aliquots were subjected to E5 and L1 PCR using BPV-1/-2
consensus primers selected from published BPV-1
(GenBank accession no. X02346) and BPV-2 (GenBank
accession no. M20219) sequences. E5-specific primers
(59B1/2-E5: 59-CACTACCTCCTGGAATGAACATTTCC39; 39B1/2-E5: 59-CTACCTTWGGTATCACATCTGGTGG39) 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 (59B1/2-L1: 59GCTAAGCAACAACAGATTCTGTTGC-39; 39B1/2-L1: 59TCAGCCATTTTGAGGTAGTCTGG-39) were designed for
amplification of a 266 bp region of the major capsid gene
L1. PCR was carried out in 0.5 ml MmlTI 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 ml DNA template in a volume of 49 ml, 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 uC 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
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cycles [92 uC for 30 s/65–56 uC for 45 s (21.5 uC per cycle)/
72 uC for 45 s], followed by 40 standard cycles (92 uC for
30 s/56 uC for 45 s/72 uC 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.
In the next step, whole blood (268 ml per individual) was
collected in Vacutainer CPT Cell Preparation Tubes with
sodium citrate (Becton Dickinson) from four sarcoidbearing horses (horses A, B, D and E), one mare suffering
from an histologically confirmed vaccination abscess
Fig. 1. (a) Optimized PCR for detection of ¡10 copies of BPV-1
E5 and L1. Top panel: E5 PCR; bottom panel: L1 PCR. Lanes 2–
10, serial 10-fold dilutions of BPV1-pML in BPV-free equine DNA;
lane 11, BPV-free equine DNA (negative control). (b, c) Detection
of E5 (b) but not L1 (c) in PBMCs from sarcoid-affected equines.
(b) E5 PCR of sarcoid-affected equines (horses A, B, D and E), a
vaccination abscess (horse C), BPV-free individuals (negative
controls, horses F, G and H), and BPV-1-positive sarcoid DNA
(positive control, horse J). ad, Sterile water control. (c) Top panel:
E5 PCR; bottom panel: L1 PCR. Horse K is a sarcoid-affected
horse, L is a horse with penile SCC and M is a horse with
periocular SCC. Horses B, C, E and F are as described in (a).
Subscripts: b, PBMCs; sa, sarcoid; ds, distant intact skin; ps,
perilesional skin; s, skin; m, melanoma; scc, squamous cell
carcinoma; va, vaccination abscess; w, juvenile wart. Lanes 1
and 12 (a) and 1 and 14 (b, c), GeneRuler 100 bp DNA Ladder
(Fermentas).
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S. Brandt and others
(horse C) and one healthy pony (horse H), the latter
serving as a PBMC-negative control. Tissue was collected
from a sarcoid of horse B, distant intact skin of sarcoidbearing horse E and from lesions of two sarcoid-free horses
F and G used as controls, comprising an equine
papillomavirus type 1-induced juvenile wart (Fw) and a
melanoma (Gm). Following PBMC isolation according to
the instructions of the manufacturer (Becton Dickinson),
blood cells and tissue specimens were subjected to DNA
extraction as described above. b-Actin PCR performed
under the conditions described above (primers 59EBA 476:
59-TCACCCACACTGTGCCCATCTACG-39; 39EBA 1090:
59-CGTCRTACTCCTGCTTGCTGATCC-39;
GenBank
accession no. AF035774) confirmed successful DNA purification for all isolates (results not shown). Subsequent E5
PCR from 2 ml DNA aliquots were positive for tumour (Bsa)
and distant skin DNA (Eds) of sarcoid-affected horses B and
E, respectively, as well as for PBMCs of sarcoid-bearing
individuals A, B, D, E and the abscess-bearing mare C.
PBMC or tissue DNA of control animals F, G and H was
negative (Fig. 1b; Table 1). Sarcoid DNA of an individual
with a determined BPV-1-infection (Jsa) was used successfully as positive control. To confirm specificity, one PBMCderived 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; Chambers et al., 2003a).
Subsequently, DNA was purified from sarcoid tissue (Ksa),
perilesional skin (Kps) and distant intact skin (Kds) of a
sarcoid-bearing horse K, from a penile squamous cell
carcinoma (SCC) from horse L (Lscc), from a periocular
SCC (horse M; Mscc) and from skin from the margin of the
vaccination-induced abscess of horse C (Cva). Following
successful b-actin PCR (not shown), DNA isolates were
screened for the presence of E5 and a 266 bp region of L1.
PBMC and tissue DNA from sarcoid-bearing individuals B
and E were included in the reaction to determine the
reproducibility of the E5 PCR results and presence of L1.
PCR detected E5 in PBMC, skin and tumour DNA of
sarcoid-affected individuals B, K and E, in tissue DNA
derived from the vaccination abscess (Cva) and also in the
periocular SCC (Mscc). Sequencing of the SCC-derived E5
amplicon revealed three point mutations compared with
laboratory clones with 97.7 % identity to the predicted
BPV-1 E5 protein sequence Swiss III. The penile SCC (Lscc)
was E5-negative, as were the negative controls (Fw, Gm and
Hb) and the no-template control (sterile water). L1 PCR
revealed 266 bp amplimers for affected and unaffected skin
of sarcoid-bearing horses (B, E and K), whereas E5-positive
DNA isolates from PBMC (Bb), abscess (Cva) and SCC
(Mscc), and the E5-negative penile SCC (Lscc), were
negative for L1 (Fig. 1c; Table 1).
Finally, blind PCRs for E5 and L1 were carried out from
enciphered PBMC DNA isolates (1–66) obtained from 66
Table 1. Sample specifications and PCR results
ND,
Not determined.
Horses
Disease
Patients
A
B
Sarcoid
Sarcoid
C
Vaccination abscess
D
E
Sarcoid
Sarcoid
K
Sarcoid
L
M
1–66
Penile SCC
Periocular SCC
Isolates 20, 24, 33: sarcoids
Others: no BPV-induced malignancies
BPV-free individuals (negative controls)
F
Juvenile warts
G
Melanomas
H
Healthy
BPV-infected individual (positive control)
J
Sarcoid
1392
Sample type
Sample code
E5 PCR
PBMC
PBMC
Sarcoid
PBMC
Abscess
PBMC
PBMC
Distant intact skin
Distant intact skin
Perilesional skin
Sarcoid
SCC
SCC
PBMC
Ab
Bb
Bsa
Cb
Cva
Db
Eb
Eds
Kds
Kps
Ksa
Lscc
Mscc
1–66
+
+
+
+
+
+
+
+
+
+
+
2
+
20+, 24+, 33+
Others
Wart
Melanoma
PBMC
Fw
Gm
Hb
2
2
2
2
2
2
Sarcoid
Jsa
+
+
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L1 PCR
ND
2
+
ND
2
ND
2
+
+
+
+
2
2
2
Journal of General Virology 89
BPV-1/-2 DNA in PBMCs of sarcoid-affected equines
co-stabled horses (donated by Monika Seltenhammer, our
laboratory). The success of DNA purification was confirmed by b-actin PCR (not shown) and photometry, the
latter revealing a mean DNA concentration of 320 ng ml21.
E5 PCR from 2 ml 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.
To our knowledge, this report is the first to demonstrate
BPV DNA in PBMCs obtained from sarcoid-affected
horses. Controls derived from virus-free individuals were
consistently negative for BPV-1/-2 E5 and L1, thus
demonstrating the absence of cross-contamination.
Sequencing performed for two of the E5 amplimers
revealed their specificity and deviation from laboratory
strains. Our findings were reproducible and strengthened
by a blind E5 PCR from PBMCs, which detected the three
sarcoid-affected horses in a cohort of 66 individuals. As
Fig. 2. A blind E5 PCR from enciphered PBMC DNAs identified
three sarcoid-bearing equines in a cohort of 66 horses. Positive
controls: sarcoid DNA Jsa and E5-containing PBMC DNA (Bb, Db
and Eb); negative controls: virus-free PBMC DNA (Fw and Hb) and
a no-template control of sterile water (ad). M, GeneRuler 100 bp
DNA ladder.
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sarcoids are mostly non-metastasizing tumours, it appears
unlikely that BPV-specific amplification products originated from sarcoid cells circulating in the blood.
Quantitative E5 PCR recently carried out by us has revealed
,100 E5 molecules per 1.26105 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/-2affected 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.
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S. Brandt and others
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 nonpermissive 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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