Detection and quantification of bovine papillomavirus DNA by digital droplet PCR in sheep blood

Abstract

Highly pathogenic bovine papillomaviruses (BPVs) 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 BPVs in 68 blood samples (66%). BPV infection by a single genotype was revealed in 61.8% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 38.2% 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 compared to conventional qPCR. Therefore, ddPCR displayed an essential diagnostic and epidemiological value very useful for 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.

Bovine delta papillomavirus E5 oncoprotein negatively regulates the cGAS-STING signaling pathway in cattle in a spontaneous model of viral disease

“…The reaction was performed in a final volume of 22 μL, containing 11 μL of ddPCR Supermix 2x for Probes, 2 μL reverse transcriptase, 1 μL DTT and 1 μL of primer and probe mix for all 4 delta BPVs. The sequences of primers and probes used to verify the presence of the delta BPV transcripts were previously used ( 22 – 24 ). The plate was transferred to an automated droplet generator (AutoDG, Bio-Rad Laboratories, Hercules, CA, USA).…”

“…BPVs are typically responsible for latent infections. BPV DNA has been found to be highly prevalent in healthy cattle ( 22 ), as well as in small ruminants that were free at pasture ( 23 , 24 ). Establishment of a persistent BPV infection is a major risk factor for cancer development and requires the virus to evade the first line of defence the innate immune system.…”

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“…In this context, our study confirmed that ddPCR can be used for low-abundance nucleic acid detection and is very useful in diagnosing infectious diseases, including viral infections in comparative medicine (29). In addition, ddPCR is very accurate and sensitive diagnostic assay for the detection and quantification of human papillomavirus DNA (25,30,31) and BPV DNA (22)(23)(24).…”

Molecular Epidemiology of Ovine Papillomavirus Infections Among Sheep in Southern Italy

“…In this context, our study confirmed that ddPCR can be used for low-abundance nucleic acid detection and is very useful in diagnosing infectious diseases, including viral infections in comparative medicine ( 29 ). In addition, ddPCR is very accurate and sensitive diagnostic assay for the detection and quantification of human papillomavirus DNA ( 25 , 30 , 31 ) and BPV DNA ( 22 – 24 ).…”

“…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 (21). Recently, ddPCR has been reported to detect and quantify bovine papillomaviruses BPVs in cattle, goats, and sheep (22)(23)(24). DdPCR has been shown to have higher accuracy than real-time quantitative PCR (qPCR).…”