Characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals

Abstract

Background: Despite the strong evidence of HPV infection as the etiological agent in a subset of oral cancer, oral α-HPV detection is rare in healthy individuals, and little is known of the existing of novel HPV types in oral cavity.

Objective: We determined whether novel HPV types can be isolated from oral rinse samples collected from healthy individuals.

Study design: We performed rolling circle amplification (RCA) coupled with degenerated PCR assay on 48 oral rinse samples to amplify novel HPV types. Full length HPV DNA was cloned using long range PCR. Quantitative type specific Taqman assays were used to determine the prevalence of novel HPV types in 158 archived oral tissue samples.

Results: We were able to isolate four novel human papillomavirus types. Full length HPV DNA was cloned for three of the four novel HPV types. All four HPV types belong to the genus Gammapapillomavirus (γ-PV), where HPV 171 is most closely related to HPV 169, showing 88% similarity; HPV 172 is most closely related to HPV 156, showing 70% similarity; HPV 173 is most closely related to HPV 4, showing 73% similarity; oral sample lavage (OSL) 37 is most closely related to HPV 144, showing 69% similarity. Finally, we showed that HPV 173 was rarely present in oral tissues (2/158), HPV 172 was only detected in normal oral tissues (25/76), and HPV 171 was more prevalent in malignant oral tissues (17/82 vs. 10/76, p=0.21).

Conclusions: Novel γ-HPV types are present in oral cavity of healthy individuals.

Keywords: HPV; Oral lavage; Oral squamous cell carcinoma.

Figure 1. Phylogenetic tree of four novel HPV types among Gammapapillomaviruses

Maximum likelihood tree was estimated by PhyML v3 . ModelTest v3.7 reported GTR+I+G substitution model as the best fit model. HPV16, an α-HPV type (underlined) was used as the out group for the tree analysis, and the tree was based on L1 sequences of 50 gamma HPV reference clones (www.hpvcenter.se). Nodes having bootstrap values less than 0.7 were labeled with an asterisk, and new HPV types identified in this study were marked by boxes.

Figure 2. HPV viral load in oral tissue samples

Formalin-fixed paraffin-embedded (FFPE) tissue blocks were selected to include both normal (oral cavity and oropharynx) and cancerous (oral cavity and oropharynx) tissues. Viral loads for three HPV types (HPV 16, HPV 171 and HPV 172) by tissue types were presented by scatter plot. The horizontal lines represent the mean viral load in each type of tissue group.