1. Introduction
Papillomaviruses (PVs) are small, non-enveloped, double-stranded DNA
viruses that infect mammals, reptiles, birds, and fish (Willemsen et
al., 2020). In mammals, PV infections have been reported in wild and
domestic, large, and small ruminants (Gibbs et al., 1975; Campo et al.,
1992; Gallina et al., 2020; Roperto et al., 2013; Roperto et al., 2016;
Savini et al., 2016). At present, 29 genotypes of bovine
papillomaviruses (BPVs) are known to infect large ruminants such as
cattle and buffaloes (Campo et al.,
1992; Roperto et al., 2013). In small ruminants, Capra hircus
papillomavirus type 1 (ChPV1) and ChPV2 are the only two caprine
genotypes responsible for PV-associated diseases in goats (Van Doorslaer
et al., 2006; Willemsen et al., 2020). Species-specific PV infections
are also known to occur in sheep. Ovine papillomaviruses (OaPVs)
comprise four members, namely OaPV1, OaPV2, OaPV3 and OaPV4. OaPV1,
OaPV2 and OaPV4 form OaPV species 3 within the genusDelta -papillomavirus, whereas OaPV3 belongs to the genusDyokappa- papillomavirus
(http://pave.niaid.nih.gov/).
OaPVs have been suggested to be associated with skin tumors (Gibbs et
al., 1975; Vanselow et al., 1982; Trenfield et al., 1990; Tilbrook et
al., 1992; Hayward et al., 1993), as ultrastructural electron-dense
particles showing papillomaviral features in symmetry and size have been
observed in cutaneous papillomas and papillomatosis of sheep (Gibbs et
al., 1975; Uzal et al., 2000). Furthermore, using cell- and
bacteria-free inocula obtained from ovine warts, an experimental
infection resulting in cutaneous proliferative lesions was transmitted
to healthy sheep (Gibbs et al., 1975). Although the complete genomes of
OaPV1 and OaPV2 have been reported a long time ago
(http://pave.niaid.nih.gov/), their actual role in the molecular
pathway involved in cutaneous and mucosal tumorigenesis of sheep remains
to be elucidated, as their association with skin tumors has been poorly
investigated in sheep (Alberti et al., 2010). OaPV3 and OaPV4 have been
recently identified in tumors of sheep from the Mediterranean region
(Sardinia Island, Italy) (Alberti et al., 2010; Tore et al., 2017). It
has been suggested that OaPV3 may represent a key factor in the pathway
of ovine cutaneous squamous cell carcinoma (SCC), as OaPV3 DNA was
detected in up to 65% of ovine SCCs (Vitiello et al., 2017).
Furthermore, OaPV4, which appears to be most closely related to OaPV1,
has been identified in sheep fibropapilloma (Tore et al., 2017). It has
been shown that E6 and E7 are the major oncoproteins through which OaPV3
and OaPV4 immortalize primary sheep keratinocytes; however, only OaPV3
displays its transforming activity through both E6 and E7 oncoproteins
(Tore et al., 2019). Ovine Delta- PVs share several biological
properties with bovine Delta -PVs, such as cell tropism, as they
can infect epithelial and mesenchymal cells (Tore et al., 2017). Similar
to bovine Delta -PV, it has been suggested that the biological
properties of ovine Delta -PV may be characterized by
cross-species transmission. OaPV2 DNA sequences have been found in a
sarcoid-like mass in the mouth of a pig (Munday et al., 2020).
Digital polymerase chain reaction (dPCR) is a new generation of PCR
techniques that enables accurate absolute quantification of target
molecules with high sensitivity. Droplet digital PCR (ddPCR) allows
massive partitioning of DNA of the sample into millions of
nanoliter-sized droplets that ideally contain either no particles or a
single particle (Kanagal-Shamanna, 2016). Recently, ddPCR has been
reported to detect and quantify bovine papillomaviruses BPVs in cattle,
goats, and sheep (Cutarelli et al., 2021; De Falco et al., 2021; Roperto
et al., 2021). DdPCR has been shown to have higher accuracy than
real-time quantitative PCR (qPCR). Therefore, ddPCR is currently the
most accurate and sensitive method for measuring the abundance of
nucleic acids of interest. DdPCR has demonstrated superior diagnostic
performance than other available molecular techniques and is very useful
in detecting low nucleic acid concentrations of oncogenic viruses,
including PVs (Biron et al., 2016). Therefore, ddPCR technology is
important in performing epidemiological investigations on the incidence
ratio of PVs and their territorial prevalence.
This study aimed to investigate OaPV detection and quantification in the
blood of apparently healthy sheep using ddPCR. In addition, the ddPCR
assay data for OaPV detection and load quantification were compared to
real-time quantitative PCR (qPCR) as qPCR is considered to be the
standard, method with the highest sensitivity and specificity for
detecting PVs DNA and cDNA (Biron et al., 2016).