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).