Abstract
A putative novel papillomavirus has been implicated in a debilitating papillomatosis–carcinomatosis syndrome affecting endangered Western barred bandicoots (Perameles bougainville ). Multiply-rimed rolling circle amplification was employed to amplify circular DNA from lesional tissue. This was subsequently used to deduce viral genetic sequences. DNA probes were generated for the L1, L2 (major and minor capsid proteins) and large T-antigen-like (putative oncoprotein) open reading frames. A genomic probe was also constructed from the entire viral genome, randomly cut into fragments. These probes were labelled by nick translation with digoxigenin and annealed probes were detected using antidigoxigenin alkaline phosphatise-conjugated F(ab)2 fragments and visualized with exposure to precipitating BM Purple alkaline phosphatase substrate solution (all reagents for in situ hybridization: Roche Diagnostics, Indianapolis, IN, USA). Endogenous alkaline phosphatase activity, which was especially prominent in apocrine sweat gland tissue, was blocked with levamisole. There was positive nuclear staining of keratinocytes and sebocytes in lesional biopsies from wart-affected Western barred bandicoots sampled between 2000 and 2006 for all DNA probes tested. Overfixation, freezing and autolysis all reduced the sensitivity and specificity of this technique; however, with optimally fixed, well-preserved tissue samples, positive staining was obvious and reliable. These results confirm that genetic sequences from a putative novel papillomavirus are situated within the histological lesions of the Western barred bandicoot papillomatosis–carcinomatosis syndrome, providing further evidence to support the hypothesis that this syndrome has a viral aetiology.