Genetic Variations of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

Viral genetic diversity within infected cells or tissues, called viral quasispecies, hasbeen mostly studied for RNA viruses, and is also described with DNA viruses includinghuman papillomavirus type 16 (HPV16) in cervical precancerous lesions. However, aprecise picture of HPV genetic variations in a cervical specimen and its involvement inHPV-induced carcinogenesis remains unclear. Here, by employing a deep sequencingtechnology, we established a novel system to comprehensively analyze geneticvariations in the full-genome HPV16 in individual clinical specimens. Throughoverlapping full-circle PCR, approximately 8-kb DNA covering the whole HPV16genome was amplified from HPV16-positive cervical exfoliated cells collected frompatients with either low-grade squamous intraepithelial lesion (LSIL) or invasivecervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novoassembly of the full-genome HPV16 sequence for each of 7 specimens (5 LSIL and 2ICC samples). Subsequent mapping of read sequences to the assembled sequencesrevealed that 2 LSIL and 1 ICC samples contained nucleotide substitutions within E6,E1 and the early poly-A sequences with mutation frequencies of 0.60% to 5.42%. Intransient replication assays, one resultant E1 mutant found in ICC, Q381E, showed areduced ability to support HPV16 origin-dependent replication. In addition, partiallydeleted sequences of E2 gene were detected in 1 LSIL sample as a mixed state withintact E2 gene. Thus, the system developed in this study provides a fundamentalframework to gain