HPV16 intratypic variants have been extensively studied. It has been proposed that HPV16 variants with E6 sequence variation are most closely related to the risk factor for development of squamous intraepithelial lesions and invasive carcinoma . In the present study, HPV16 prototype (18.3%) and the 5 different HPV16 variant sub-lineages, As (61%), AA1 (8.5%), EUR (7.3%), AFR2 (3.7%) and J135C (1.2%), were detected (Table 1). The HPV16As detection rate increased according to the severity of the lesion, 30% of LSIL, 63.9% of HSIL and 66.7% of SCC. This result shows that HPV16As infection is associated with a high risk of HSIL and SCC development with an odds ratio of 4.387; 95% CI 1.043-18.457; P = 0.042 compared to prototype and other sub-lineages. Moreover, in comparison to HPV16 prototype, this result shows an increased association of HPV16As with risk for cervical cancer. This study suggests that HPV16As is an oncogenic risk for cervical cancer progression.
A study of HPV16 variants in Khon Kaen, Thailand found HPV16As in 73.9% of HPV16-positive cervical cancer samples and showed a risk association with CIN II-III and SCC . Our previous and present studies confirmed the strong association of HPV16As with cervical cancer development in Thai women. Some studies have shown that infection with HPV16 prototype is associated with a lower risk in progression to cervical cancer than that caused by other variants. Sequence variation among HPV16 variants may influence the event of HPV persistence and progression to CIN and cervical carcinoma [9, 15].
Using the HPV16 prototype [GenBank:AY686584] as a reference sequence, we detected a total of 38 nucleotide variation positions in the LCRs of 43 HPV16As cases. This result agrees with previous reports of a HPV16As-specific nucleotide variation in the LCR at position 7842 [16–18]. At this position (7842), the majority of the LCR samples had a nucleotide change from G>A (90.7%), whereas the remaining samples had a G>T change. We also identified additional nucleotide variations at positions 7175A>C, 7177 T>C, 7193 G>T, 7201 T>C, 7287A>C, 7521 G>A, 7730A>C, 24C>T and 81 G>T, which were found in all (100%) samples. Other common variations were 7270C>T (95.3%) and 7289 A>T (95.3%). These sequence variations may be typical of the LCR from HPV16As in this region (Table 2).
In this study, 10 novel nucleotide variations, which were previously unreported in the literature, were found in HPV16As LCRs (Table 2); however, two of them were found in only one sample (7617C>A and 7844A>C) and may have occurred by PCR amplification. These variations were associated with YY-1 binding sites, and among them, a deletion or substitution was found to enhance early promoter transcription. It was suggested that mutation affecting YY1-motifs in the LCR is one of the mechanisms that enhance viral oncogene expression during the course of cancer cell progression . Additionally, several studies have reported that cellular factors, such as AP-1, GRE, NF-1, NF-IL6, OCT-1, SP-1, TEF-1, TEF-2 and YY-1, either stimulate or inhibit p97 promoter activity [20–22]. Therefore, these variations could be related to the early promoter activation of HPV16As. With respect to positions 7429 G>A, 7874C>G, 28 G insertion with A, 46 T insertion with G and 61 T insertion with G, these mutations are located close to E2BS-4 (nt 7453–7464), E2BS-3 (nt 7860–7871), SP-1 and E2BS-2 (nt 35–46) and E2BS-1 (nt 50–61), respectively . These novel nucleotide variations in the LCR of HPV16As may play a crucial role in the transcriptional modulation of the HPV16 E6 and E7 oncogenes via the p97 promoter. Lace et al.  reported that p97 promoter activity of CAT reporter containing different LCR mutation in HeLa cell line. The results showed that transcriptional activity of HPV16As LCR variations was higher than that of the HPV16 prototype.
The HPV16As isolated from samples no. 15 (HSIL case) and 36 (SCC case) with common nucleotide variation in the LCR (Tables 2 and 3) showed an approximate 5-fold increase in p97 promoter activity compared to the prototype. In accordance with previous results , EUR and AA1 transcriptional activity studied in the present study also show higher activity than prototype. This study also shows higher activity of EUR than common HPV16As.
Interestingly, the activity of AA1 has similar patterns to the LCR from the HPV16As-sv1 and HPV16As-sv14 which contained novel variations proximal to the p97 promoter that showed transcriptional activity with 19 and 30-fold higher activity than the prototype. These data suggest that novel nucleotide changes at 7429 G>A and 7874C>G, which are proximal to p97 (at positions 28, 46 and 61) in the LCRs from HPV16As, are associated with high transcriptional activity. Therefore, the oncogenic potential of HPV16As could be influenced by specific sequence variation, especially at positions that are proximal to the promoter region in the HPV16 LCR.
Veress et al.  reported transcriptional activity of LCRs from AA HPV16 variants by luciferase assay in C33A cells. The results showed a 1.7-fold increase in transcriptional activity with the AA isolate LCR and a very similar transcriptional activity with the EUR LCR compared to that with the LCR reference. This increased activity of the AA isolate could be attributed to nucleotide changes found at the 3’ end of the LCR (nt7660-7890).
In addition, in 2001 Veress et al.  showed that the transcriptional activity of HPV16 full-length AA and EUR isolated from clinical specimens was higher than prototype.
Kammer et al.  reported that 3.3- and 2.8-fold increases in p97 promoter activity were detected in the Asian American c (AAc) and North American 1 (NA-1) variants, respectively, when compared with the European reference clone. The region in the AAc and NA-1 variants that is responsible for enhanced transcription could be the E6-proximal end of the LCR (nt 7619–124). Similar results were obtained by Veress et al. , who showed that the enhanced transcriptional activity of the AAc variant was due to nucleotide changes in the 3’ region of the LCR. However, a deletion variant lacking the whole enhancer and both silencer regions, either the YY-1-specific silencer alone or together with the CDP-specific silencer, retained substantial enhancer activity on the p97 promoter. Chen et al.  studied mutations in the LCR and their functional consequences in oral cancer. They found that promoter activity of the mutated HPV16 LCR was significantly higher than that of the wild type HPV16 LCR, suggesting that mutations in the LCR of HPV in oral cancer leads to increased expression of the E6/E7 oncoproteins, which might contribute to the carcinogenic process.