Human Papillomavirus (HPV) infection is clearly established as the cause of cervical cancer. A distinct cause and effect relationship of infection to cancer does not, however, exist, since the majority of infections are self-limiting, benign, and never progress to cancer. A large body of molecular research has uncovered many of the viral mechanisms believed responsible for later development of cancer, but to date has not resolved why progression to cancer takes many years to occur, if it occurs at all. This, in part, has hindered development of assays capable of predicting which patients will progress to more severe disease. The most widely used assay for HPV is the solution-based DMA hybrid capture assay (HCA). Several recent epidemiological studies have clearly shown that HCA has a strong negative predictive value; the ability to confidently state that a HPV-negative patient will not develop cervical cancer over the next several years. HCA also does not have high positive predictive value, in part due to the inability to differentiate between normal productive infection, and cells containing integrated viral DNA, which are found in dysplasia and cervical cancer. Using a novel probe labeling method, this Small Business Innovation Research project proposes to develop a sensitive in situ hybridization assay for detecting RNAs of high-risk HPV viral types. This SBIR will validate the assay using in vitro skin reconstruct models as a surrogate model for clinical HPV infections. ? ?