Ovine papillomaviruses (OaPVs) were detected and quantified, for the first time, using droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (qPCR) via liquid biopsy of 165 clinically healthy sheep. OaPV DNA was detected in 126 blood samples (~76.4%). DdPCR detected OaPV DNA in 124 samples; in only two additional samples positive for real-time qPCR, ddPCR failed to detect the presence of any OaPVs. In 70 of the positive samples (~55.6%), a single OaPV infection was observed, 12 of which were caused by OaPV1 (~17.1%) and 14 by OaPV2 (20%). OaPV3 was responsible for 19 single infections (~27.1%), and OaPV4 for 25 single infections (~35.7%). Multiple OaPV coinfections were observed in 56 (~44.4%) positive samples. OaPV coinfections caused by two genotypes were observed in 31 positive samples (~55.4%), with dual OaPV3/OaPV4 infection being the most prevalent as seen in 11 blood samples. In addition, five OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, four OaPV1/OaPV3, and three OaPV2/OaPV4 dual coinfections were also detected. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~42.8%) and only one (~1.8%) of coinfected blood samples, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent, as observed in 12 (50%) blood samples harboring triple OaPV infections. This study showed that ddPCR is the most sensitive and accurate assay for OaPV detection and quantification thus outperforming real-time qPCR in terms of sensitivity and specificity. Therefore, ddPCR may represent the molecular diagnostic tool of choice, ultimately providing useful insights into OaPV molecular epidemiology and field surveillance.

Ovine papillomavirus (OaPV) comprises four genotypes; OaPV1, OaPV2, and OaPV4 are fibropapillomaviruses within the genus Delta-papillomavirus ( Delta-PV ), whereas OaPV3 is an epitheliotropic virus that belongs to the genus Dyokappa-papillomavirus ( Dyokappa-PV ). To date, all of them have been known to infect sheep only. OaPV1, OaPV2, and OaPV4 have been associated with ovine cutaneous and mucosal fibropapillomas, while OaPV3 is a key factor in the squamous cell carcinoma (SCC) pathway of the sheep skin. Peripheral blood mononuclear cell (PBMC) samples obtained from 128 cattle at public slaughterhouses were investigated using droplet digital polymerase chain reaction (ddPCR). ddPCR is a new-generation PCR technique that enables accurate and absolute quantification of target molecules with high sensitivity and specificity. All OaPVs were detected by identification and quantification of nucleic acids using specific fluorescent probes. Of 128 PBMC samples, 100 (~78%) showed OaPV infections. Further, 42, 35, and 23 PBMC samples showed single, double, and triple OaPV infections, respectively. OaPV1 was responsible for 22 single infections, OaPV2 caused 16 single infections, and OaPV3 and OaPV4 caused two single infections each. OaPV1 and OaPV2 were the most frequent ovine viruses in dual and triple infections. In many PBMC samples, both ovine Delta-PV and Dyokappa-PV were found to be transcriptionally active, as shown by the detection and quantification of E5 oncogene transcripts for OaPV1, L1 transcripts for OaPV2, E6 and E7 transcripts for OaPV3, and E6 for OaPV4. OaPVs were found in the blood samples from cattle that shared grasslands rich in bracken ferns known to contain immunosuppressant substances. Furthermore, OaPVs were also found in cattle from intensive livestock farming without any contact with sheep. Because OaPV DNA was detected in both grass hay and corn silage, it is conceivable that these feed may be the viral sources.

Persistent infection and tumourigenesis by papillomaviruses (PVs) require viral manipulation of various of cellular processes, including those involved in innate immune responses. Herein, we showed that bovine PV (BPV) E5 oncoprotein interacts with a tripartite motif-containing 25 (TRIM25) but not with Riplet in spontaneous BPV infection of urothelial cells of cattle. Statistically significant reduced protein levels of TRIM25, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were detected by Western blot analysis. Real-time quantitative PCR revealed marked transcriptional downregulation of RIG-I and MDA5 in E5-expressing cells compared with healthy urothelial cells. Mitochondrial antiviral signalling (MAVS) protein expression did not vary significantly between diseased and healthy cells. Co-immunoprecipitation studies showed that MAVS interacted with a protein network composed of Sec13, which is a positive regulator of MAVS-mediated RLR antiviral signalling, phosphorylated TANK binding kinase 1 (TBK1), and phosphorylated interferon regulatory factor 3 (IRF3). Immunoblotting revealed significantly low expression levels of Sec13 in BPV-infected cells. Low levels of Sec13 resulted in a weaker host antiviral immune response, as it attenuates MAVS-mediated IRF3 activation. Furthermore, western blot analysis revealed significantly reduced expression levels of pTBK1, which plays an essential role in the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. Our results suggested that the innate immune signalling pathway mediated by RIG-I-like receptors (RLRs) was impaired in cells infected with BPVs. Therefore, an effective immune response is not elicited against these viruses, which facilitates persistent viral infection.

Abstract – Highly pathogenic bovine Delta papillomaviruses (δPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected bovine δPVs in 68 blood samples (66%). Bovine papillomavirus (BPV) infection by a single genotype was revealed in 59% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 41% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection. Therefore, ddPCR displayed diagnostic and epidemiological value resulting in the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.

In the present study, the highly pathogenic bovine Deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real time PCR (qPCR). Overall, ddPCR detected BPV E5 DNA in ~61.3% of the blood samples examined, while real time qPCR revealed the virus in ~10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR revealed a single BPV genotype in ~59.2% and multiple genotypes in ~40.8% of goats harboring BPV DNA, while real time qPCR detected single genotypes in ~17% and multiple genotypes in ~1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes, and six (~14%) four genotypes. In contrast, real time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that the ddPCR technique has much higher sensitivity and specificity in the detection of these viruses, and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.

In this study, the digital droplet polymerase chain reaction (ddPCR) was used to quantify circulating bovine papillomavirus (BPV; genus: Deltapapillomavirus) levels in blood samples from 25 healthy cows and 15 cows with chronic enzootic hematuria due to papillomavirus-associated bladder tumors. ddPCR detected the BPV DNA in 95% of all the samples (i.e., in 24 of the healthy cows and 14 of the diseased animals), whereas quantitative real time PCR (qPCR) detected it in only 57.5% of the same blood samples, with the percentage differences between ddPCR and qPCR being statistically significant (P value  0.05), according to the 2 test of Campbell and Richardson. Furthermore, ddPCR detected BPV infections by a single genotype and by multiple genotypes in 37% and 63% of the cows, respectively, whereas qPCR detected these in 16% and 16%, respectively. Of the two assays, ddPCR was the more sensitive and accurate clinical diagnostic tool, allowing the detection of otherwise undetectable BPV genotypes, and consequently, a higher number of BPV co-infections. qPCR failed to detect many BPV co-infections by multiple genotypes. Therefore, ddPCR may be an essential tool for improving diagnostic procedures, allowing the identification of the genotypic distribution of BPV and a better understanding about the territorial divergence, if any, of the BPV prevalence in different areas. No significant differences in the blood viral load estimations were observed between the two animal groups, suggesting that the bloodstream could be a site of primary infection. Finally, as BPV DNA was detected in cows affected by noninvasive urothelial tumors, including papilloma and papillary urothelial neoplasms of low malignant potential, the circulating BPVs appeared to be independent of the status of urothelial neoplasms. Therefore, unlike in humans, circulating BPVs cannot be an actual prognostic marker of urothelial tumors in cows.