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Citation: Maggi, R.; De Paolis, L.; De
Santis, D.; Vellone, V.G.; De Ciucis,
C.G.; Fruscione, F.; Mazzocco, K.;
Ghelardi, A.; Marruchella, G.;
Razzuoli, E. Bovine Papillomavirus
Type 1 Infection in an Equine
Congenital Papilloma. Pathogens
2023,12, 1059. https://doi.org/
10.3390/pathogens12081059
Academic Editors: Laura Gallina
and Magda Dunowska
Received: 17 April 2023
Revised: 14 August 2023
Accepted: 15 August 2023
Published: 18 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pathogens
Case Report
Bovine Papillomavirus Type 1 Infection in an Equine
Congenital Papilloma
Raffaella Maggi 1, Livia De Paolis 2, Daria De Santis 3, Valerio Gaetano Vellone 4, Chiara Grazia De Ciucis 2,
Floriana Fruscione 2, Katia Mazzocco 4, Alessandro Ghelardi 5, Giuseppe Marruchella 6, *
and Elisabetta Razzuoli 2, *
1Veterinary Practitioner, Via Cassia 829, 00189 Rome, Italy; raffy.maggi@hotmail.it
2Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, National Reference Center of
Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila 29/34, 16129 Genova, Italy;
livia.depaolis@izsto.it (L.D.P.); chiaragrazia.deciucis@izsto.it (C.G.D.C.); floriana.fruscione@izsto.it (F.F.)
3Veterinary Practitioner, Via San Manno 19, 03024 Cepranno, Italy; dariadesantisvet@gmail.com
4U.O.C. Anatomia Patologica, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy;
valeriovellone@gaslini.org (V.G.V.); katiamazzocco@gaslini.org (K.M.)
5Azienda Usl Toscana-Ovest, UOC Ostetricia e Ginecologia, Ospedale Apuane, 54100 Massa, Italy;
ghelardi.alessandro@gmail.com
6Faculty of Veterinary Medicine, University of Teramo, 64100 Teramo, Italy
*Correspondence: gmarruchella@unite.it (G.M.); elisabetta.razzuoli@izsto.it (E.R.);
Tel.: +39-086-126-6932 (G.M.); +39-010-542-274 (E.R.)
Abstract:
Papillomas are benign epithelial lesions protruding on the epithelial surfaces as finger-like
or warty projections. These lesions are often caused by papillomavirus (PV) infection. Congenital
papillomas have been reported in foals. However, to date, no evidence of PV infection has been
provided. In the present paper, we describe the main clinical–pathological features of a congenital
papilloma observed in a foal. In addition, biomolecular tests demonstrated BPV1 infection in the
case under study. Such data stimulate further investigations, even on archived samples, aiming to
clarifying the etiology of equine congenital papilloma and the clinical relevance, if any, of BPV1
vertical transmission in horses.
Keywords: horse; congenital papilloma; bovine papillomaviruses
1. Introduction
Papillomas are benign epithelial lesions protruding from the epithelial surfaces as
finger-like or warty projections [
1
]. Papillomas are usually caused by papillomaviruses
(PVs, fam. Papillomaviridae), non-enveloped and host-adapted viruses provided with a
circular dsDNA genome [
2
]. Microscopically, cutaneous papillomas consist of arborizing
fibrovascular fronds lined with a thickened epidermis, more prominent within the stratum
spinosum. Large, variably shaped keratohyalin granules, and degenerating cells (so called
koilocytes) are commonly observed. Moreover, a few intranuclear viral inclusions can be
detected throughout the stratum spinosum and granulosum [3,4].
As far as the horse is concerned, three syndromes caused by Equus caballus papillo-
maviruses (EcPVs) have been described:
•
classic equine viral papillomatosis via EcPV-1, which usually affects the muzzle and
lips of young horses (<3 years of age) and regresses spontaneously within 2–3 months;
•
equine genital papillomas caused by EcPV-2, which affect older horses and do not
resolve spontaneously;
•
equine ear papillomas caused by EcPV3-6, which present with bilateral and symmet-
ric white hyperkeratotic plaques confined to the pinnae, and rarely resolve sponta-
neously [3].
Pathogens 2023,12, 1059. https://doi.org/10.3390/pathogens12081059 https://www.mdpi.com/journal/pathogens
Pathogens 2023,12, 1059 2 of 9
In addition, equids develop sarcoids after bovine PV (BPV) type 1 and 2 infection, this
being among the very few cases of cross-species PV infection reported so far. Sarcoids are
locally invasive fibroblastic tumors, and they represent the most common and economically
relevant neoplasms in horses [3,5].
Congenital papillomas have also been reported in foals. To date, no evidence of
EcPV infection has been provided, suggesting that equine congenital papillomas might be
regarded as epidermal hamartomas [
6
,
7
]. The present paper describes the main clinical–
pathological features of a congenital papilloma recently observed in a foal. The presence of
PV infection was also investigated.
2. Case Description
In March 2022, a male, newborn quarter horse foal underwent a routine clinical
examination. The foal was born on his due date, and appeared healthy, bright, and alert.
However, a spheric, pedunculated, and non-painful mass was observed, which hung from
the upper lip and did not prevent suckling (Figure 1).
Pathogens 2023, 12, x FOR PEER REVIEW 2 of 10
In addition, equids develop sarcoids after bovine PV (BPV) type 1 and 2 infection,
this being among the very few cases of cross-species PV infection reported so far. Sarcoids
are locally invasive fibroblastic tumors, and they represent the most common and eco-
nomically relevant neoplasms in horses [3,5].
Congenital papillomas have also been reported in foals. To date, no evidence of EcPV
infection has been provided, suggesting that equine congenital papillomas might be re-
garded as epidermal hamartomas [6,7]. The present paper describes the main clinical–
pathological features of a congenital papilloma recently observed in a foal. The presence
of PV infection was also investigated.
2. Case Description
In March 2022, a male, newborn quarter horse foal underwent a routine clinical ex-
amination. The foal was born on his due date, and appeared healthy, bright, and alert.
However, a spheric, pedunculated, and non-painful mass was observed, which hung from
the upper lip and did not prevent suckling (Figure 1).
Figure 1. Foal three days after birth. A globoid lesion is clearly seen, aached to the upper lip.
The mass was surgically removed three days later. To achieve this, the foal was drug-
free restrained [8], 1 mL of 2% lidocaine chlorydrate was locally injected around the base
of the peduncle, and the mass was fully removed and promptly fixed in 10% neutral-buff-
ered formalin (Figure 2). The skin incision healed without any complication, and the
stiches were removed 10 days after surgery.
Figure 1. Foal three days after birth. A globoid lesion is clearly seen, attached to the upper lip.
The mass was surgically removed three days later. To achieve this, the foal was drug-
free restrained [
8
], 1 mL of 2% lidocaine chlorydrate was locally injected around the base of
the peduncle, and the mass was fully removed and promptly fixed in 10% neutral-buffered
formalin (Figure 2). The skin incision healed without any complication, and the stiches
were removed 10 days after surgery.
Formalin-fixed samples were embedded in paraffin and routinely processed in prepa-
ration for histopathological examination (hematoxylin and eosin stain, H and E). Micro-
scopically, the mass consisted of several finger-like fibro-vascular projections, which were
covered by a thickened epidermis with prominent acanthosis and orthokeratotic hyperker-
atosis (Figure 3). We observed evidence of koilocytosis, keratohyalin granule abnormalities,
and a pilosebaceous unit (Figure 4).
Pathogens 2023,12, 1059 3 of 9
Pathogens 2023, 12, x FOR PEER REVIEW 3 of 10
(a) (b)
Figure 2. Excised lesion after formalin fixation. The mass is about 3 cm in diameter and shows a
verrucous, “truffle-like” surface (a). In the cut section, the arborized stromal scaffold can be clearly
observed (b).
Formalin-fixed samples were embedded in paraffin and routinely processed in prep-
aration for histopathological examination (hematoxylin and eosin stain, H and E). Micro-
scopically, the mass consisted of several finger-like fibro-vascular projections, which were
covered by a thickened epidermis with prominent acanthosis and orthokeratotic hyper-
keratosis (Figure 3). We observed evidence of koilocytosis, keratohyalin granule abnor-
malities, and a pilosebaceous unit (Figure 4).
Based on the clinical history, gross findings, and histopathological features, the diag-
nosis of equine congenital papilloma was made.
Figure 3. Histopathological examination of the excised lesion. In this finger-like projection, a central
fibrovascular backbone is clearly seen, which contains several blood and lymphatic vessels. The
stroma is covered by a thickened epidermis, the stratum spinosum being markedly expanded. H
and E stain. Final magnification ×200.
Figure 2.
Excised lesion after formalin fixation. The mass is about 3 cm in diameter and shows a
verrucous, “truffle-like” surface (
a
). In the cut section, the arborized stromal scaffold can be clearly
observed (b).
Pathogens 2023, 12, x FOR PEER REVIEW 3 of 10
(a) (b)
Figure 2. Excised lesion after formalin fixation. The mass is about 3 cm in diameter and shows a
verrucous, “truffle-like” surface (a). In the cut section, the arborized stromal scaffold can be clearly
observed (b).
Formalin-fixed samples were embedded in paraffin and routinely processed in prep-
aration for histopathological examination (hematoxylin and eosin stain, H and E). Micro-
scopically, the mass consisted of several finger-like fibro-vascular projections, which were
covered by a thickened epidermis with prominent acanthosis and orthokeratotic hyper-
keratosis (Figure 3). We observed evidence of koilocytosis, keratohyalin granule abnor-
malities, and a pilosebaceous unit (Figure 4).
Based on the clinical history, gross findings, and histopathological features, the diag-
nosis of equine congenital papilloma was made.
Figure 3. Histopathological examination of the excised lesion. In this finger-like projection, a central
fibrovascular backbone is clearly seen, which contains several blood and lymphatic vessels. The
stroma is covered by a thickened epidermis, the stratum spinosum being markedly expanded. H
and E stain. Final magnification ×200.
Figure 3.
Histopathological examination of the excised lesion. In this finger-like projection, a central
fibrovascular backbone is clearly seen, which contains several blood and lymphatic vessels. The
stroma is covered by a thickened epidermis, the stratum spinosum being markedly expanded. H and
E stain. Final magnification ×200.
Based on the clinical history, gross findings, and histopathological features, the diag-
nosis of equine congenital papilloma was made.
Total DNA was extracted from three 5
µ
m thick sections of formalin-fixed and paraffin-
embedded (FFPE) samples using the AllPrep DNA FFPE kit (Qiagen, Milan, Italy), accord-
ing to the manufacturer’s instruction. The DNA concentration was measured using the
Qubit 3 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA). Aiming to investigate
the presence of the EcPV2/9/10 and BPV1/2/13 genome, 100 ng of DNA, 200 nM of
the probe, and 100 nM of each primer were added to 20
µ
L of the iTaq Universal Probes
Supermix (BioRad, Milan, Italy), in a total volume of 25
µ
L. Equine beta-2-microglobulin
(
β
2M) was used as the gene reference. Specific primer and probe sequences are reported in
Pathogens 2023,12, 1059 4 of 9
Table 1. Internal controls (block blanks, extraction blanks, and positive controls) were used
for each analytical session. Real-time PCR was performed using a CFX96 Real-Time System
(BioRad, Milan, Italy). A threshold cycle of 38 was set as the cut off for sample positivity.
All samples were tested in duplicate.
Pathogens 2023, 12, x FOR PEER REVIEW 4 of 10
Figure 4. In these sections, evidence of koilocytosis was observed. The right panels reveal a close-
up view of the inset. The arrows indicate some selected koilocytes. H and E stain. Final magnifica-
tion ×200.
Total DNA was extracted from three 5 µm thick sections of formalin-fixed and par-
affin-embedded (FFPE) samples using the AllPrep DNA FFPE kit (Qiagen, Milan, Italy),
according to the manufacturer’s instruction. The DNA concentration was measured using
the Qubit 3 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA). Aiming to inves-
tigate the presence of the EcPV2/9/10 and BPV1/2/13 genome, 100 ng of DNA, 200 nM of
the probe, and 100 nM of each primer were added to 20 µL of the iTaq Universal Probes
Supermix (BioRad, Milan, Italy), in a total volume of 25 µL. Equine beta-2-microglobulin
(β2M) was used as the gene reference. Specific primer and probe sequences are reported
in Table 1. Internal controls (block blanks, extraction blanks, and positive controls) were
used for each analytical session. Real-time PCR was performed using a CFX96 Real-Time
System (BioRad, Milan, Italy). A threshold cycle of 38 was set as the cut off for sample
positivity. All samples were tested in duplicate.
Table 1. Primer set and probes used in the virological investigations.
Target Gene Primer Sequences Amplicon
Length
Accession Num-
ber
EcPV2-L1 F-5′-TTGTCCAGGAGAGGGGTTAG-3′ 80 NC_012123.1
R-5′-TGCCTTCCTTTTCTTGGTGG-3′
p-EcPV2-L1 FAM-CGTCCAGCACCTTCGACCACCA-TAMRA
EcPV9-L1 F-5′-TTC ATC CCA GCT TGA GAC CA-3′ 116 MN117918.1 R-5′-GCA GAT CAA TGG TCC AGA AGG-3′
p-EcPV9-L1 p-FAM-ATT GCC TCC TCA GCC ACC CG-TAMRA
EcPV10-L1 F-5′-GTG TCA CAG GTA ACC CCC TG-3′ 174 OP870083 R-5′-AAG CGT GTC TTC CTC CAG TG-3′
p-EcPV10-L1 p-FAM-TGC TGG TGG GTT GCA AGC CC-TAMRA
BPV1-L1 F-5′-CAG GAC TGT TCA CAA CCC AA-3′ 96 NC_001522.1
R-5′-CCC AGT TAC AGT ACC TCC AA-3′
p-BPV1-L1 p-FAM-TGC AGG TGT CCA GAG GGC AG-TAMRA
BPV2-L1 F-5′-ACA GCC CGT CCA TGT GTT A-3′ 115 MF045490
R-5′-TCA GCA GCA CCA AAC CCT AT-3′
Figure 4.
In these sections, evidence of koilocytosis was observed. The right panels reveal a close-up
view of the inset. The arrows indicate some selected koilocytes. H and E stain. Final magnification
×200.
Table 1. Primer set and probes used in the virological investigations.
Target Gene Primer Sequences Amplicon Length Accession Number
EcPV2-L1 F-50-TTGTCCAGGAGAGGGGTTAG-30
80 NC_012123.1
R-50-TGCCTTCCTTTTCTTGGTGG-30
p-EcPV2-L1 FAM-CGTCCAGCACCTTCGACCACCA-TAMRA
EcPV9-L1 F-50-TTC ATC CCA GCT TGA GAC CA-30
116 MN117918.1
R-50-GCA GAT CAA TGG TCC AGA AGG-30
p-EcPV9-L1 p-FAM-ATT GCC TCC TCA GCC ACC CG-TAMRA
EcPV10-L1 F-50-GTG TCA CAG GTA ACC CCC TG-30
174 OP870083
R-50-AAG CGT GTC TTC CTC CAG TG-30
p-EcPV10-L1 p-FAM-TGC TGG TGG GTT GCA AGC CC-TAMRA
BPV1-L1 F-50-CAG GAC TGT TCA CAA CCC AA-30
96 NC_001522.1
R-50-CCC AGT TAC AGT ACC TCC AA-30
p-BPV1-L1 p-FAM-TGC AGG TGT CCA GAG GGC AG-TAMRA
BPV2-L1 F-50-ACA GCC CGT CCA TGT GTT A-30
115 MF045490
R-50-TCA GCA GCA CCA AAC CCT AT-30
p-BPV2-L1 p-FAM-AGA AAA TGG TGC GTG TCC TCC T-TAMRA
BPV1-E5 F-50-TGCTTCAATGCAACTGCTGCT-30
77 MZ310894
R-50-AGGAGCACTCAAAATGATCCCAG-30
p-BPV1-E5 p-FAM-ACTCTTGTTTTTTCTTGTA-TAMRA
BPV1-E6 F-50-TGCTACTGTGGGGGCAAACT-30
110 MZ310894
R-50-CAGTCGTAGCAGCGTCCTCT-30
p-BPV1-E6 p-FAM-AGCCTTTCTGCAAAACCAGAGCT-TAMRA
BPV1-E7 F-50-GCTGTGGAAACTGCGGAAAA-30
122 MZ310894
R-50-GCGAGATTCACAACGTGGAC-30
p-BPV1-E7 p-FAM-GCTGACTTTTGCAGTGAAGACCAGC-TAMRA
BPV13-L1 F-50-GCA CCC CAC TTT TAA TGC CT-30
87 NC_030795
R-50-TCC TGT TTG CTT CCT GTC ATC-30
p-BPV13-L1 p-FAM-AGG AAA GTG ACC AGC CAA ACA ACA-TAMRA
B2M (Beta2-microglobulin) F-50-GGCTACTCTCCCTGACTGG-30
135 NM_001082502.3
R-50-TCAATCTCAGGCGGATGGAA-30
p-B2M p-FAM-ACTCACGTCACCCAGCAGAGA-TAMRA
Pathogens 2023,12, 1059 5 of 9
Real-time PCR demonstrated the presence of the BPV1 genome, while it gave a nega-
tive result for all the other PVs herein investigated (see Table 2and Figure S1 for details).
Table 2.
Real-time PCR targeting the PV genome. Data are expressed as Cq mean value
±
stan-
dard deviation.
Sample Real-Time PCR
B2M EcPV2-L1 EcPV9-L1 EcPV10-L1 BPV2-L1 BPV13-L1
29.4 ±0.1 >38 >38 >38 >38 >38
B2M BPV1-E5 BPV1-E6 BPV1-E7 BPV1-L1
29.1 ±0.1 21.5 ±0.2 21.7 ±0.4 21.5 ±0.2 22.5 ±0.5
Thereafter, to evaluate the expression of BPV1-L1 and oncogenes (namely, E5, E6, E7,
and L1), total RNA was extracted from three 5
µ
m thick sections of the FFPE samples
using the Maxwell
®
RSC RNA FFPE kit (Promega, Madison, WI, USA), according to the
manufacturer’s instructions. RNA concentration was evaluated using the Qubit 3 fluo-
rimeter (Thermo Fisher, Milan, Italy). The reverse transcription (RT) step was performed
using the Reliance Select cDNA Synthesis Kit (BioRad, Milan, Italy), adding 100 ng of
RNA. Then,
5µL
of 1:3 diluted complementary DNA (cDNA) was added to 20
µ
L PCR
mixture at the final concentration of 1
×
master mix (iTaq Universal Probes Supermix,
Bio-Rad, Irvine, CA, USA), with the following thermal profile: 95
◦
C for 10
0
, then 39 cycles
of
95 ◦C
for 15
00
and 60
◦
C for 60
00
in a CFX96
™
Real-Time System. To exclude genomic
DNA contamination, a digestion using the DNase I RNase free kit (Qiagen, Milan, Italy)
was performed. The specific primer set and probes used for testing the expression of
BPV1 oncogenes are reported in Table 1. RT-Real-Time PCR demonstrated that BPV1 L1
(Cq = 37.1
±
0.3), E5 (
Cq = 30.1 ±0.2
), E6 (Cq = 34.2
±
0.2), and E7 (Cq = 33.8
±
0.3) were
expressed at the mRNA level (Table 3).
Table 3. BPV1 oncogene expression. Data are expressed as Cq mean value ±standard deviation.
Sample Real-Time PCR
B2M BPV1-E5 BPV1-E6 BPV1-E7 BPV1-L1
31.4 ±0.2 30.1 ±0.2 34.2 ±0.2 21.5 ±0.2 37.1 ±0.3
After this test, to evaluate the mRNA localization, RNA in situ hybridization was
performed using the RNAscope kit (Advanced Cell Diagnostics Inc., Hayward, CA, USA),
according to the manufacturer’s guidelines, as described in [
9
]. The RNAscope probe used
was the V-BPV-E-bovine-papillomavirus-1 complete genome E5 E6 E7 (Catalog Number
416831) and was designed to detect the mRNA expression of the E5, E6, and E7 genes
(National Center for Biotechnology Information Reference Sequence NC_001522.1). FFPE
tissue sections (of 5
µ
m thickness) were deparaffinized in xylene, and were subsequently
dehydrated in 100% ethanol. Tissue sections were exposed to hydrogen peroxide for 10 min,
followed by incubation in a target retrieval reagent maintained at a boiling temperature
(98 to 102
◦
C) using a hot plate for 15 min, rinsed in deionized water, and immediately
treated with Protease Plus at 40
◦
C for 30 min in a hybridization oven (TopBrite- Resnova,
RM, Italy). The tissue sections were then incubated at 40
◦
C with a target probe of BPV-1
for 2 h. The target–probe hybridization was followed by a series of target-specific signal-
amplification steps. After each hybridization step, slides were washed with wash buffer
at room temperature twice, followed by staining with Fast Red dye. The sections were
then counterstained with 50% hematoxylin staining solution, and dehydrated using xylene,
before being mounted with a mounting medium (Eukitt). The positive signals were present
in the form of punctate cytoplasmic and nuclear red staining that was higher than the signal
on the negative control slide. Assays using FFPE specimens were performed in parallel
Pathogens 2023,12, 1059 6 of 9
with positive and negative control probes (positive probe: Ec-PPIB; negative control probe:
DapB), to ensure interpretable results (Figure 5).
Pathogens 2023, 12, x FOR PEER REVIEW 6 of 10
was the V-BPV-E-bovine-papillomavirus-1 complete genome E5 E6 E7 (Catalog Number
416831) and was designed to detect the mRNA expression of the E5, E6, and E7 genes
(National Center for Biotechnology Information Reference Sequence NC_001522.1). FFPE
tissue sections (of 5 µm thickness) were deparaffinized in xylene, and were subsequently
dehydrated in 100% ethanol. Tissue sections were exposed to hydrogen peroxide for 10
min, followed by incubation in a target retrieval reagent maintained at a boiling tempera-
ture (98 to 102 °C) using a hot plate for 15 min, rinsed in deionized water, and immediately
treated with Protease Plus at 40 °C for 30 min in a hybridization oven (TopBrite- Resnova,
RM, Italy). The tissue sections were then incubated at 40 °C with a target probe of BPV-1
for 2 h. The target–probe hybridization was followed by a series of target-specific signal-
amplification steps. After each hybridization step, slides were washed with wash buffer
at room temperature twice, followed by staining with Fast Red dye. The sections were
then counterstained with 50% hematoxylin staining solution, and dehydrated using xy-
lene, before being mounted with a mounting medium (Euki). The positive signals were
present in the form of punctate cytoplasmic and nuclear red staining that was higher than
the signal on the negative control slide. Assays using FFPE specimens were performed in
parallel with positive and negative control probes (positive probe: Ec-PPIB; negative con-
trol probe: DapB), to ensure interpretable results (Figure 5).
Figure 5. RNAscope assay. (A) Tissue sections hybridized with the probe targeting BPV-1
E5-E6-E7 mRNA (V-BPV-E). A close-up view of the inset is visible in the right panel. Spe-
cific staining was observed in the congenital papilloma under investigation. In detail, scat-
tered red dots were detected within the cytoplasms (empty circles) and nuclei (black ar-
row) of epithelial cells. (B) On the contrary, no specific staining was observed in the neg-
ative control. Final magnification: ×400.
3. Discussion
Papillomaviruses are regarded as very important oncogenic viruses, able to induce
benign and often self-limiting epithelial neoplasms, which can occasionally progress to
malignancy [10,11]. In humans, cervical cancer represents the most common PV-associ-
ated cancer [12]. Moreover, the role of human PVs as causative agents of anogenital, head,
Figure 5.
RNAscope assay. (
A
) Tissue sections hybridized with the probe targeting BPV-1 E5-E6-E7
mRNA (V-BPV-E). A close-up view of the inset is visible in the right panel. Specific staining was
observed in the congenital papilloma under investigation. In detail, scattered red dots were detected
within the cytoplasms (empty circles) and nuclei (black arrow) of epithelial cells. (
B
) On the contrary,
no specific staining was observed in the negative control. Final magnification: ×400.
3. Discussion
Papillomaviruses are regarded as very important oncogenic viruses, able to induce
benign and often self-limiting epithelial neoplasms, which can occasionally progress to
malignancy [
10
,
11
]. In humans, cervical cancer represents the most common PV-associated
cancer [
12
]. Moreover, the role of human PVs as causative agents of anogenital, head, and
neck cancers is widely accepted [
13
]. Likewise, PVs have been associated with malignant
neoplasms in several animal species [
11
]. In horses, a growing body of evidence suggests
that EcPV2 infection contributes to the etiopathogenesis of squamous cell carcinomas,
which can affect the external genitalia, and laryngeal and gastric mucosa [
14
,
15
]. Other
types of equine papillomaviruses have been associated with different lesions. In particular,
papillomas seem to be caused by EcPV1, EcPV3-6, and EcPV8, while EcPV9 and EcPV10, to
date, are not associated with specific pathology [16–18].
Papillomaviruses are strictly species-specific and show a distinct tropism towards
epithelial cells. Delta-PVs (e.g., BPV1 and BPV2) are exceptions to this general rule, as they
have a wider host range and can infect different cell types [
11
,
19
]. A recent case report by
Savini and co-workers (2020) demonstrated the ability of BPV1 to cause proliferative lesions
in sheep. These results suggest a different pathogenetic role of BPV-1, and confirm the
ability of BPV1 to infect different species, also causing a non-sarcoid outcome on cutaneous
surfaces [
20
]. Notably, phylogenetic investigations revealed that BPV1 originated in cattle,
while multiple cross-species transmissions occurred into horses [
21
]. The causal association
of BPV1 and BPV2 infection with equid sarcoid has been unequivocally demonstrated [
22
].
It was assumed that BPV1/2 infections were strictly confined to dermal fibroblasts in the
Pathogens 2023,12, 1059 7 of 9
horses, wherein they reside in a non-productive episomal form. Consequently, horses have
been long considered a dead-end host for BPV1/2 [
14
]. However, BPV1 infection has been
demonstrated to involve the epidermis of the horse, where it may be productive, albeit at
low level [
23
]. In addition, BPV1 can early-infect PBMCs in young horses, likely inducing a
viremic phase [
24
], and the BPV1 genome has been detected in the placenta and blood of
newborn foals [25].
Overall, biomolecular investigations clearly demonstrated BPV1 infection in the equine
congenital papilloma under study. As a matter of fact, the detection of BPV1 oncogene
mRNA and its cytoplasmatic localization ruled out any incidental contamination at the time
of sampling and/or during laboratory tests. Moreover, according to the literature, such data
would be unlikely to result from the selective infection of PBMCs, considering the high viral
load detected (the BPV1 DNA was 100-fold more concentrated than the equine genome,
when compared with the
β
2M), as well as the transcription of the L1 gene [
24
,
26
,
27
].
The present case report is apparently in contrast with those currently available in the
literature [
6
,
7
]. However, we remark that White et al. [
6
] and Postey et al. [
7
] only ruled
out the presence of EcPVs in equine congenital papillomas, while no data have ever been
provided about BPV1 infection in such lesions.
Although PV infections mainly spread through horizontal transmission, a growing in-
terest exists around intrauterine and perinatal transmission. In humans, the PV (human PV,
HPV) genome has been detected in the amniotic fluid, placenta, and umbilical cord
[28,29]
,
and transplacental infection might occur through the hematogenous or ascending route,
from the maternal genital tract [
30
]. The hypothesis of vertical transmission has been further
corroborated in cattle, as BPV2 was shown to productively infect the uterine epithelium and
chorionic placenta [
31
,
32
]. Accordingly, some evidence supports BPV vertical transmission
in sheep [
33
]. The mechanism of BPV transmission in horses is still debated; the BPV
genome has been detected in the equine placenta, and intrauterine transmission has been
speculated in this animal species [
25
]. Moreover, Silva and co-workers demonstrated the
virus’ presence in the PBMC and semen of healthy horses [
27
,
34
]. Therefore, recently, it
was speculated that, as in humans, infection could be ascribable to oocyte fecundation [
35
].
Considering this, the detection of BPV1 in congenital papillomas argues in favor of its
transplacental transmission in horses, stimulating further investigation to identify the risk
factors, prevalence, and clinical relevance of such event.
Congenital papillomas have been repeatedly reported in animals (including horses) and
regarded as hamartomatous lesions, with no viral origin having been demonstrated
[1,6,7]
. It
is worth noting that Roperto et al. [
33
] recently reported BPV-associated congenital lesions
in lambs after transplacental infection. Of course, the conclusive demonstration of the viral
etiology (if any) of equine congenital papilloma goes well beyond the scope of the present
case report. Moreover, the molecular detection of BPV1 in newborn healthy foals [
25
],
along with the conflicting data about BPV1 infection in the skin of healthy horses [
36
,
37
],
add further concerns about the significance of BPV1 in equine congenital papilloma. In
our opinion, the case described herein suggests that BPV vertical transmission might have
clinical relevance in different animal species, further highlighting the biological plasticity
of delta-PVs. As a matter of fact, BPV1/2 have been shown to infect several animal species,
targeting a wide range of cell types and causing different neoplastic lesions depending on
the host features (animal species, age, route of transmission).
4. Conclusions
The present case report found BPV1 infection in an equine congenital papilloma, with
very high viral load. This stimulates further investigations, even on archived samples,
aiming to clarify the etiology of equine congenital papilloma and the clinical relevance, if
any, of BPV1 vertical transmission in horses.
Pathogens 2023,12, 1059 8 of 9
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pathogens12081059/s1, Figure S1: Curve of Real Time PCR to
detection of BPV1.
Author Contributions:
Conceptualization, G.M., R.M., A.G. and E.R.; methodology, G.M., R.M., F.F.,
D.D.S. and E.R.; formal analysis, G.M., L.D.P., C.G.D.C., F.F., V.G.V. and K.M.; investigation, G.M.,
R.M., L.D.P., C.G.D.C. and E.R.; resources, G.M. and E.R.; data curation, V.G.V., K.M., D.D.S., G.M.,
R.M. and E.R.; writing—original draft preparation, D.D.S., G.M., R.M. and E.R.; writing—review and
editing, V.G.V. and K.M.; visualization, G.M., A.G. and E.R.; supervision, G.M., A.G. and E.R.; project
administration, E.R.; funding acquisition, E.R. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the Italian Ministry of Health and the Liguria Region, grant
number IZS PLV 12/19 and 22L03. The APC was funded by IZS PLV 12/19 and 22L03.
Institutional Review Board Statement:
The study was approved by the Ethics Committee of Is-
tituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta (protocol code 14047 of
28 November 2019).
Informed Consent Statement:
Informed consent was obtained from the owner of the horse involved
in this study.
Data Availability Statement: All data deriving from the study are given in the article text.
Conflicts of Interest: The authors declare no conflict of interest.
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