1. Introduction
Papillomaviruses (PVs) are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of animals in a species-specific manner (IARC, 2007). Bovine papillomaviruses (BPVs) consist of 28 genotypes that are distributed into five genera: Deltapapillomavirus (δPV) ( BPV-1, -2, -13, and -14), Xipapillomavirus (χPV) (BPV-3, -4, -6, -9, -10, -11, -12, -15, -17, -20, -23, -24, -26, and -28),Epsilonpapillomavirus (εPV) (BPV-5, -8, and -25),Dyokappapapillomavirus (DyoκPV) (BPV-16, -18, -22),Dyoxipapillomavirus (DyoχPV) (BPV-7). BPV-19, -21, and -27 remain to be classified (http://pave.niaid.nih.gov/; Crespo et al., 2019 ).
Bovine δPV is generally considered to be associated with a high risk of neoplastic transformation (Daudt et al., 2018). Indeed, this BPV genus plays a central role in both cutaneous and bladder tumors of large ruminants (Campo et al., 1992; Roperto et al., 2013). In southern Italy, BPV-2 and -13 are the most important infectious agents involved in bladder carcinogenesis in Podolica breed cattle (Roperto et al., 2010; Roperto et al., 2016a).
Although bovine δPVs recognize Bos taurus as their classical host, this virus genus is the only one known to be characterized by natural cross-species transmission and infection (IARC, 2007). Bovine δPVs cause cutaneous tumors in horses (Lancaster et al., 1979), buffaloes (Silvestre et al., 2009; Pangty et al., 2010; Somvanshi, 2011), cats (Munday et al., 2015), African lions (Orbell et al., 2011), and Cape mountain zebras, giraffes and sable antelopes (Williams et al., 2011). Bovine δPV DNA has been identified in a squamous cell carcinoma involving the head and neck of a mare and in cutaneous wart lesions from sheep (Kainzbauer et al., 2012; Mazzucchelli-de-Souza et al., 2018).
The small-sized (40-85 amino acids) E5 protein, believed to be the major oncoprotein of δPV, displays cell-transforming activity via numerous pathways (DiMaio and Petti, 2013). The E6 protein of δPVs appears to contribute to cell transformation through its interaction with the focal adhesion protein paxillin. With regard to the E7 protein, although it has no cell-transforming functionality by itself, the transformation process appears to be correlated with the protein’s ability to bind to p600.
Furthermore, PV infections are significantly associated with many adverse effects on the reproductive function, which may be related to the infecting genotype (Souho et al., 2015). A recent viral metagenomics analysis revealed a high prevalence of BPV-1 and -2 in the uterus of apparently heathy dairy cows (Ling et al., 2019). Furthermore, BPV DNA has been found in the uterus of healthy mares (Savini et al., 2019). BPVs are able to cause a congenital infection via transplacental transmission in cows and buffaloes (Roperto et al., 2019; Russo et al., 2020). Transplacental infections of BPV-2 and -13 in ewes have also been reported (Roperto et al., 2018).
There are few reports presenting quantification data of BPVs, and those that do are based on quantitative real-rime polymerase chain reaction (qPCR) analysis. Indeed, qPCR has for a long time been the method of choice for viral load estimation (Lillsunde Larsson and Helenius, 2017) and is considered the assay with the highest sensitivity and specificity for the detection of PV DNA and cDNA (Biron et al., 2016). To date, BPV-1 and -2 are the only genotypes that have been investigated by qPCR (Yuan et al., 2007; Cota et al., 2015).
The droplet digital polymerase chain reaction (ddPCR) is a robust PCR technique that is currently the most accurate and sensitive method for quantifying nucleic acids of interest. Unlike that by qPCR, quantifcation by ddPCR is absolute and does not require calibration of standards, thus rendering the process faster, more precise, and reproducible (Kanagal-Shamanna, 2017). Although ddPCR has not yet been reported for the detection of BPV, it is believed to have a potential higher accuracy than qPCR for detecting the virus. Sensitive and specific biomarkers for the blood-based analysis of analytes, such as viral DNA, as well as accurate molecular epidemiological tools, are still needed to achieve a superior diagnostic performance in identifying BPVs.
As there are currently no published studies on the quantification of circulating bovine δPVs, the aim of the present study was to evaluate the performance of ddPCR in quantifying the viral load of high-risk genotypes (1, 2, 13, 14) of circulating bovine δPV. The same blood samples were simultaneously assayed by qPCR in order to compare the diagnostic sensitivity of the two assays for this virus.