Localization of Bovine Papillomavirus Nucleic Acid in Equine Sarcoids

Abstract

Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids.

Bovine Papillomavirus DNA and S100 Profiles in Sarcoids and Other Cutaneous Spindle Cell Tumors in Horses

“…1,2,7,12,16,25,26,35 Although BPV DNA is found in various locations within sarcoids, it also can be found in normal skin areas of horses with sarcoids as well as within skin of horses with dermatitis. 2,6,17,18,37 Sarcoid induction and other tumorigenic effects that might be relevant to the more widespread association of BPV-1/2 DNA with equine spindle cell tumors indicated by this study have been linked most closely with cellular expression of E5 protein. BPV-1 is capable of inducing proliferation and transformation in cultured dermal fibroblasts from horses, mice, and humans.…”

Papillomaviruses: a systematic review

“…The association between BPV and equine sarcoid was first described by Olson and Cook in 1951 (Brandt et al , 2008). BPV-1 and 2 DNA sequences are identified in 100% of equine sarcoids (Martens et al , 2001a; Gaynor et al , 2015b). Furthermore, DNA sequences of these virus types are identified in 2/3 of asymptomatic horses (Bravo et al , 2010).…”

Dermatopathology – the link between ancillary techniques and clinical lesions

“…Variably labelled small complementary DNA or RNA probes can be applied to fresh snap‐frozen tissues, FFPE tissues, smears or microarrays. The complementary molecular probes are either radiolabelled or conjugated to fluorescent compounds (fluorescence in situ hybridization; FISH), horseradish peroxidase or digoxigenin . The detection system depends on the label used [digoxigenin: 4‐nitrobIue tetrazolium chloride (NBT)/15‐bromo‐4‐chloro‐ 3‐indolyl‐phosphate (BCIP); fluorophore: light of appropriate wavelength; horseradish peroxidase‐based signal: using a chromogenic such as 3,30‐diaminobenzidine (DAB).…”

“…Moreover, it is important to prove causality of a particular pathogen identified by PCR. This often requires application of additional techniques such as immunohistology or in situ hybridization . On the other hand, false negative results may occur due to low quality DNA/RNA in a sample.…”

“…In tissue sections, the positive results can be visualized in the context of the morphological lesions. Hence, ISH avoids false positive results as can be seen with PCR . Moreover, ISH and IHC can be applied simultaneously on a single sample .…”