Molecular detection of Ovis aries Papillomavirus type 3 in formalin fixed, paraffin embedded (FFPE) sheep squamous cell carcinoma samples

Recently (Alberti et al., 2010) the prototype of a novel papillomavirus genus (OaPV3) was detected in normal skin and squamous cell carcinoma (SCC) lesions of Sardinian sheep. OaPV3 belongs to the Dyokappa genus and its genome significantly differs from the 2 species of ovine papillomaviruses previously reported in Australia that instead group with the artiodactyl deltapapillomavirus species, and have been isolated from benign cutaneous lesions. Here we investigate the relevance of OaPV3 in SCC by screning a panel of FFPE sheep SCC samples. Based on the sequence of OaPV3 L1 and E6 two DNA probes and a set of 4 primers were designed and respectively used to develop an In situ Hybridisation test (ISH) and two RT-PCR assays. These assays were applied to a collection of 41 FFPE sheep SCCs samples obtains from cutaneous tumours (5 nasal, 6 ear, 10 periocular, 3 dorsal, and 17 mammary tumours). Diagnosis of SCC was confirmed by histopathological examination of the 41 samples. Molecular tests, summarised in the table, revealed that 26 out of 41 (63%) samples were positive to at least one test. DifFerent tests showed difFerent sensitivites. Also, tumours localised in different parts of the sheep body seemed to show variable degrees of positivity to OaPV3. Results demonstrate a high prevalence (63%) of OaPV3 in sheep SCs. This level of prevalence is particularly important and comparable to the prevalence of HPVs in human SCC (50 to 69%, Meyer et al.,2000), and suggests that OaPV3 represent an important risk factor for the development of sheep SCC. The level of positivity of nasal and periocular lesions was greater respect to other tumour locations. This can be explained by the greater level of solar expositon and/or to traumas of these area respect to other locations. As expected, RT-PCR has a greater sensitvity than ISH. However, only combining these two tests the total number of positives can be detected, and both the presence/expression and localisation in the tumour can be investigated. Concluding, we cannot rule out the presence of unknown viral types in negative tissues. Further investigation is needed to investigate the presence of viral variants associated to different tumour locations and the presence of uncovered papillomaviruses in negative samples.