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.