Papillomavirus Detection by Partial or Complete Genome Sequencing

Objective: Head and neck (H&N) cancer is the sixth leading cause of cancer in the US. Papillomavirus (PV) infection of susceptible mucosal lined surfaces of the oral cavity is associated with benign and malignant tumor development. Detection of human papillomavirus (HPV) in the oral cavity increases cancer risk by 15 to 200-fold. Coupled with clinical and histological data, PV detection can assist in disease classification, and has been used to forecast response to treatment in H&N cancer. Our objectives were to 1) highlight an effective PCR-based technique for HPV detection and, 2) describe the method for full-genome analysis of a canine PV subtype.

Method: DNA was isolated from paraffin-embedded labial mucosa and lip samples from the University of Louisville Oral Pathology Laboratory (LOPL). Examination of the clinical and histological records of the two patients suggested focal epithelial hyperplasia (FEH). The universal HPV primers My09/My11 were used for PCR amplification and the resulting products were sequenced.  Separately, DNA was isolated from one frozen-tissue sample from a canine anogenital biopsy. Subsequent amplification was accomplished by the multiply primed rolling-circle technique to obtain the viral plasmid. The circular DNA was linearized and cloned into a pUC19 vector. All genomic and phylogenetic comparison was completed using the NCBI BLAST database and DNAStar software.

Result: Samples from the LOPL harbored HPV32, supporting the diagnosis of FEH. Whole viral genome isolation from the canine anogenital biopsy indicated the presence of canine oral papillomavirus (COPV).

Conclusion: The universal HPV primers My09/My11 were used to confirm a diagnosis of FEH in two patients. Furthermore, using whole genome purification and analysis we indicate a novel anogenital tropism for COPV. These two approaches for PV identification emphasize the relative simplicity of PV detection and highlight the power of molecular biology techniques to inform the researcher and clinician.