Identification and characterization of novel human papillomaviruses in oral rinse samples.
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Abstract Background With close to 200 different human papillomavirus (HPV) types in the papilloma virus genome database (http://pave.niaid.nih.gov), it is not surprising that there are still uncharacterized novel HPVs present in the oral cavity and oropharynx. In our previous study we discovered and fully cloned three novel types of HPVs in healthy patients (Martin et al., J Clinical Virology. 2014 Jan:59(1):30). We hypothesize that there are new, as yet unidentified oncogenic HPVs present in the oral cavity and oropharynx of head and neck cancer patients, which could be identified using next generation sequencing (NGS) technology. Objectives The objectives of this study were to: i) Discover novel HPVs using Next Generation Sequencing (NGS) technology in oral rinse samples collected from oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) patients; ii) Determine prevalence of novel HPVs in archived OSCC/OPSC tissue samples; and iii) Examine frequency of novel oncogenic HPVs in cancer and non-cancer oral rinse samples using real-time PCR. Methods We collected 110 oral rinse samples from healthy patients and 100 oral rinse samples from patients with OSCC/OPSCC. Enrichment of HPV DNA was completed using multiply-primed rolling-circular amplification (MP-RCA) techniques. Fluorescent arbitrarily primed (FAP) PCR methods were used to isolate the L1 region of potential novel HPVs. NGS was used to detect for HPVs from 7-pooled samples that consisted of samples that underwent enrichment and FAP PCR. Potential novel HPVs were identified through cloning and Sanger sequencing methods. BLASTn and PaVE databases were used for nucleotide searches. Phylogenetic trees were created to determine related HPVs and genus. New primers and probes were created for the novel HPVs in order to test prevalence in 221 archived tissue biopsies and 210 oral rinse samples. Results We discovered three potential novel HPVs: NV14.4, NV69.1, and NV95. NV14.4 has 89% homology to HPV76; NV69.1 has 85% homology to HPV152; and NV95 has 77% homology to HPV147. From the archived tissue biopsy samples, only 0.8% of the OSCC patients were positive for NV14.4; NV69.1 and NV95 were not detected in the samples. Of the OPSCC oral rinse samples: 1% was positive for NV14.4; 13% was positive for NV69.1; and 1% was positive for NV95. Of the OSCC oral rinse samples: 6% was positive for NV14.4; 12.5% was positive for NV69.1; and 6% was positive for NV95. Of the other head and neck cancer oral rinse samples 12.5% was positive for NV69.1; NV14.4 and NV95 were not detected. None of the non-cancer samples in the tissue biopsy set and the oral rinse sample set were positive for the three novel HPVs. Conclusions Novel, potentially oncogenic, HPVs can be detected in oral rinse samples using NGS technology in conjunction with cloning and Sanger sequencing.
- Dentistry