Production andin vitromanipulation of oncogenic animal papillomaviruses

Papillomaviruses are a diverse group of small, non-enveloped, double stranded DNA viruses that cause proliferations of the stratified squamous epithelium of the skin and mucosa in a wide variety of vertebrate hosts. Papillomaviruses play an important role in human cancer development, and have been isolated from a number of animal malignancies. This project focuses on the recently described ovine papillomavirus OaPV3, a virus associated with squamous cell carcinoma (SCC) in sheep. Like all papillomaviruses, OaPV3 is characterised by the inability to productively infect replicating cells in culture, this hampering studies on infection, biological cycle, and isolation. Therefore, we generated OaPV3-based non-infectious virus-like particles (VLPs) through expression of the viral L1 major capsid protein in insect cells, using recombinant baculoviruses. Also, infectious OaPV3 pseudovirions (PsVs) have been produced, that can transduce reporter genes in 293TT cells.VLPs were used to develop an ELISA test for measuring the presence of antibodies in immunized and naturally infected sheep, as well as to produce sera and monoclonal antibodies useful for detecting OaPV antigens in direct diagnosis and as specific assay controls. OaPV3 pseudovirions were also produced and used in infection and neutralisation assays with sera and monoclonal antibodies raised against VLPs, to investigate the potential of OaPV3 virus-like particles as vaccines candidates.