The results showed that high risk- (HR) HPVs were detected in 37/189 (19.6%) PCa specimens compared to 16/167 (9.6%) of BHP specimens . These data suggest HR-HPV may play a role in PCa. HPV 52 and 58 were the most frequent genotypes  detected in the population studied. Koilocytes were detected in all in situ PCR-HPV-positive samples, representing a pathognomonic feature of infection, and we observed the overexpression of p16-INK4A in HPV-positive samples compared to HPV-negative samples, indirectly suggesting the presence of HR-HPV E7 oncoprotein. These results suggest that HPV infection plays an important role in prostate cancer development.The aim of this study was to evaluate the association between prostate cancer (PCa) and Human papillomavirus (HPV) infection in the Mexican population. We studied 356 paraffin-embedded tissues from unrelated Mexican men with PCa or benign prostatic hyperplasia (BPH), with the latter serving as control. HPV detection was performed by polymerase chain reaction (PCR) using universal primers, and viral genotypes were detected using sequencing or multiplex PCR. Light microscopy analyses enabled the identification of koilocytes in samples subsequently analyzed for HPV detection by  Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of death from cancer in men (Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections (STIs) worldwide .HPV infection is also one of the causes of intraprostatic inflammation, and there is evidence showing that chronic inflammation is involved in the regulation of cellular events in prostate carcinogenesis  tissue samples, and the case group comprised 189 tissue samples from men diagnosed with PCa, which was confirmed by histological analysis. The Institutional Human Research Ethical Committee approved the protocol.DNA was extracted from paraffin-embedded tissue samples using the DNeasy Blood and Tissue Kit  according to the manufacturer’s protocol. DNA concentrations were spectrophotometrically determined at 260 nm. The integrity of the DNA samples was assessed by electrophoresis in 1% agarose gels, with the human beta-globin gene being amplified by polymerase chain reaction (PCR) as internal control. HPV detection was performed using consensus primers to amplify part of the L1 gene HPV region, following the previously demonstrated efficacy of PCR amplification from a variety of genital HPV types (All HPV-positive tissue samples were stained with hematoxylin and eosin (HE). Briefly, the tissues were incubated with Harris’ hematoxylin for 15 min and subsequently washed with distilled water, followed by acid alcohol, running water, and 2% sodium bicarbonate. Subsequently, the samples were fixed in 80% ethanol for 2 min, placed in alcoholic eosin solution for 10 min, and then the samples were decolorized with 90% ethanol to remove excess dye. Finally, the samples were analyzed using a light field optical microscope at 20× magnification.in situ PCR was performed using E6/E7 specific primers, as previously described . The amplification of DNA was accomplished using a hot start method with two consensus sequence primer pairs within E6 and E7 of high-risk HPV (pU-1M and pU-2R primers) . The cycling conditions were 2 min at 94 ºC and 18 cycles of 94 ºC for 1 min, 60 ºC for 1 min and 72 ºC for 1 min. Clips and AmpliCover discs were removed and the slides were washed in PBS, followed by 5 min in 100% EtOH prior to air drying.To identify the high-risk HPV (HR-HPV)  E6/E7 viral genes, ications . Brieflyications . NegativWe used an indirect immunolabeling method with a primary anti-digoxigenin antibody  conjugated to alkaline phosphatase to detect the PCR product. Briefly, blocking was performed in 5% BSA  in PBS for 30 min. The slides were subsequently drained and an anti-DIG antibody  was applied (100 mL per sample) for 2 h at room temperature. The detection of alkaline phosphatase was performed for 10 min using an NBT/BCIP kit (Roche). After detection, the slides were rinsed in distilled water for 5 min and counterstained with Fast Green. The slides were air-dried and subsequently mounted in Permount histological mounting medium .Images were obtained using a DFC290 HD digital camera . The image files were opened with PhotoImpact software  and digitally processed to obtain a more homogeneous signal.Sections of 5 μm in thickness were obtained from the paraffin blocks and mounted on electrocharged slides. Subsequently, the tissues were de-paraffinized and rehydrated as previously described , as a measure of the association between HPV infection and the risk of PCa.p=0.01).In this study, we examined the presence of HPV in paraffin-embedded tissue samples from unrelated Mexican men with PCa or BPH. Using three pairs of degenerate primers targeting the L1 late gene, HPV was detected in 14.9% (53/356) of all tissues analyzed, showing higher levels in tissues with PCa (37/189 or 19.6%) than in tissues with BPH (16/167 or 9.6%), suggesting that HPV infection could be a risk factor for PCa , which is much higher than the occurrence of the low-risk (LR) HPV genotypes at 19% (17% in the BPH group and 21% in the PCa group). The viral genotypes observed in the samples in order of decreasing prevalence were HPV 52 (33.3%), HPV 58 (17.17%), HPV 11 (12.7%), HPV 18 (10.8%), HPV 16 (7.8%), HPV 33 (6.9%), HPV 6 (5.9%), and HPV 31 (4.0%) . The detWith respect to HPV genotype distribution in the study groups, in HPV infections involving a single genotype, HPV 52 was the most common genotype found in both groups, while in HPV infections with multiple genotypes, the most common genotypes were HPV 58 and 52 for the PCa group, and HPV 52, followed by HPV 11 and 58, for the control group . The mosAdditionally, we identified koilocytes, cells containing an acentric, hyperchromatic nucleus displaced by a large perinuclear vacuole. Although the genesis of the cytoplasmic vacuole remained unclear, particularly because both HPV DNA replication and virion assembly exclusively occur in the nucleus, in clinical biopsies from cervical cells, koilocytosis is observed in both LR and HR HPV infections . MoreoveIn this study, we examined the presence of HPV in two study groups: a control group comprising BPH tissues and a case group comprising cancerous tissues. We found HPV in 53 (14.9%) of the 356 tissues analyzed, which is similar to the findings in other reports of tissue analysis from Latin America (18.63%)  development of diagnostic assays; (ii) evaluation of the impact of vaccination in cancer prevention strategies, especially since many HPV genotypes are not covered by the current quadrivalent HPV vaccine used in Mexico; and (iii) description of highly prevalent genitourinary tract HPV infections in sexually active men in México (In conclusion, the high frequency of detection of HPV in PCa, combinations of genotypes with oncogenic potential that dominated in the PCa group, identification of HPV associated koilocytes, and overexpression of p16INK4A in prostate cancer specimens constitute evidence suggesting the association of HPV with PCa and a potential role for the virus in the etiology of PCa.


Human papillomavirus is recognized as a major cause of cervical cancer. It is estimated that annually, 7,095 women are diagnosed with cervical cancer and 4,732 die from the disease in Ethiopia. Understanding that the screening practice is very poor and the coverage is very limited, this disease burden is one of the major public health agendas in Ethiopia. This study aimed to assess the burden and genotype distribution of high-risk human papillomavirus (HR HPV) infection and cervical cytology abnormalities at selected obstetrics and gynecology clinics of Addis Ababa, Ethiopia.An institutional-based cross-sectional study design was employed from June to October 2015. Cervical samples were collected from 366 participants based on inclusion criteria. HR HPV DNA was analyzed using an Abbott Real-Time PCR system, and cervical cytology screening was performed using the conventional Pap-smear technique. Data were entered in to Epi-data version 13 and analyzed using STATA version 11.P = 0.01).The overall HR HPV burden and abnormal cytology were 13.7 and 13.1%, respectively. The majority of HR HPV types were other than types 16 and 18. Of the total abnormal cytology results, 81.3% were low-grade squamous intraepithelial lesions (LSILs), and 12.5 and 6.3% were atypical squamous cells of undetermined significance (ASCUS) and high-grade squamous intraepithelial lesions (HSILs), respectively. Residence, occupation, and HIV serostatus were significantly associated with HR HPV infection. Among the variables, age, age at first marriage, and education were the only ones associated with cervical cytology abnormalities. The overall agreement between the real-time PCR and Pap cytology screening methods was 78.96% , Non-16/18 HR HPV genotypes represented the largest proportion of HR HPV infections in this study. Women without cervical cytology abnormalities had the highest frequency of HR HPV infection. A large-scale community-based cohort study shall be designed and implemented to further identifying the persistent genotype and assessing the changes in cervical epithelial cell lines.The online version of this article (10.1186/s12885-019-5953-1) contains supplementary material, which is available to authorized users. The World Health Organization estimates that nearly 530,000 women worldwide are diagnosed with cervical cancer every year and that 275,000 die from the disease. Cervical cancer is renowned as the third most common cause of cancer in women globally, of which almost 70% occurs in developing countries , 2. In Ehuman papillomavirus (HPV) genotypes. The association between HPV and cervical cancer is a universal fact, and variability among the different types is geographically limited. With optimal testing systems, HPV DNA can be identified in almost all specimens of invasive cervical cancer, and infection of the cervix with HPV is the main cause of cervical cancer [Cervical cancer has been recognized as an unusual outcome of a sexually transmitted infection, and the etiology is limited to a few l cancer . One of l cancer .Of all HPV genotypes, more than 40 have been identified from anogenital mucosa samples and most are transmitted sexually. HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 are classified as the high-risk (HR) group, which predicts cervical cancer [In sub-Saharan Africa, HPV-associated cervical cancer is one of the major causes of morbidity and mortality. A lack of strong initiatives as well as sustainable cervical cancer prevention programs and services have been identified as potential causes of the high incidence rate in most countries . In EastAn institutional-based cross-sectional study design was employed at three selected obstetrics and gynecology clinics of Addis Ababa, Ethiopia, from June to October 2015. The study was conducted among women who visited the Family Guidance Association of Ethiopia Addis Ababa Area Reproductive Health Clinic, Hemen Maternal and Children Health Specialty Center, and SinamokshEthio Women’s Health Special Clinic. The study population consisted of women who visited the clinics for any gynecological purposes, including cervical cancer screening, and fulfilled the inclusion criteria. A nonprobability convenience sampling technique was used to select the study sites, considering the scope and volume of services provided. As these health facilities provide cervical cancer screening services and have a significant volume of client visits, they were potential sites for this study and among the very few sites providing this service consistently in the city. All women who visited each clinic during the study period and who were eligible for this study were consecutively added until the number of clients reached the calculated minimum sample size. A total of 366 women were enrolled in the study.P-value of less than 0.05 was considered statistically significant.Sociodemographic characteristics, sexual behaviors and other risk-factor variable responses were gathered using a structured questionnaire , were within the range of 31–60 years. In terms of residence, 352 (96.2%) participants visited the study clinics from the Addis Ababa area. Of the total number of participants, 287 (78.4%) were married; among these, 71 (24.7%) was married for the first time before 18 years of age. Regarding parity, 29 (7.9%) of the participants had > 5 complete pregnancies and deliveries; 281 (76.8%) women were parity 1 to 5.Participant employment status was also assessed, and 248 (67.8%) of the study participants were self-employed. Regarding educational status, the highest proportion comprised those with Diploma or Degree and above qualification , and only 39 (10.7%) were unable to read and write . Among the HR HPV-positive cases, 8 (16%) were identified as having HR HPV 16 genotype, 38 (76%) had “other HR HPV” , 2 (4%) had genotype 16 together with “other HR HPV” genotypes, 1 (2%) had genotype 18 together with “other HR HPV” genotypes, and 1 (2%) had genotype 18. The HR HPV genotype distribution showed that “other HR HPV” types dominated over genotypes 16 and 18 ), which was statistically significant.P = 0.000), parity (P = 0.017), age at first marriage (P = 0.027), education (P = 0.003), condom use during sexual intercourse (0.011), cigarette smoking (0.000), and family history of cervical cancer (0.003) were significantly associated with HR HPV infection. Ever use of any type of contraceptive, age at first sexual intercourse, more than one lifetime sexual partnership, history of STD, alcohol consumption, and HIV serostatus, with P-values of 0.106, 0.266, 0.334, 0.824, 0.227, and 0.688, were not significantly associated.The association between HR HPV infection with sociodemographic and reproductive health, sexual behavior, and other risk factors was analyzed through bivariate analysis using the chi-square test. Age .Overall, Pap smear abnormalities were observed in 13.1% 48/366) of the study subjects. Among the abnormalities, 3 (6.3%), 39 (81.3%), and 6 12.5%) were ASCUS, LSIL, and HSIL, respectively .Ever use of any type of contraceptive, oral contraceptive use, age at first sexual intercourse, more than one lifetime sexual partnership, frequency of condom use, frequency of cigarette smoking, history of STD, and alcohol consumption were not significantly associated with abnormal Pap cytology . The overall percent agreement findings reveal significant agreement between HR HPV DNA PCR and conventional Pap smear cytology screening methods (P < 0.05).Agreement between HR HPV DNA PCR and conventional Pap smear cervical cancer screening methods was analyzed using positive, negative, and overall percent agreement and the Kappa statistic. The positive and negative percent agreement was found to be 87.7 and 22.4%, respectively. However, the overall percent agreement was 79.0%, and the Kappa value was 0.12 (95% CI (0.00–0.24), This study mainly aimed to assess the burden of HR HPV and cervical cytology abnormalities, along with potentially associated sociodemographic, sexual behavior, and reproductive health variables, in three Obstetrics and Gynecology and reproductive health clinics in Addis Ababa, Ethiopia. In this study, the overall HR HPV burden was 13.7%. “Other HR HPV” genotypes  were the most frequent (76%) genotypes identified in this study, followed by HR HPV 16 (16%). The overall prevalence of abnormal cytology was also 13.1%. Approximately three-fourth (72%) of the HR HPV-infected women were in the age range of 31 to 60 years, and this was significantly associated with abnormal cytology. HR HPV was found in 12.6% of normal cytology reports. Moreover, residence, occupation, and HIV serostatus were significantly associated with HR HPV infection.In this study, the overall HR HPV burden was 13.7%, a finding that was consistent with previous studies reported from different parts of Ethiopia , 15, at To date, studies conducted in Ethiopia , 16 haveMultiple HR HPV type infections were found in 7.9% of HR HPV-positive individuals in a study by Mohammed et al. in Northeastern Nigeria , which iP < 0.05) was not significant in the multivariate analysis. This is similar to the results of the study conducted in Gurage Zone, Ethiopia [P < 0.0001), with a peak in the age range of 21 to 25 years. This might be due to the detection of low-risk HPV in addition to HR HPV.An age-specific HPV infection study in South Africa  reportedEthiopia . In contIn this study, residence, occupation, and HIV serostatus were significantly associated with HR HPV infection in multivariate analysis. This finding was comparable with a study  reportinIn our study, ever use of any type of contraceptive, age at first sexual intercourse, and more than one lifetime sexual partnership were not associated with HR HPV infection. This outcome is comparable to the findings of Mega AC et al. in rural Nigeria .p-value 0.004 and 0.014), consistent with a study reported by Abel et al. [The overall abnormal cytology burden in the present study was 13.1%, which was lower than that in a similar study from Ethiopia , 22 and Furthermore, our study presents high-risk HPV genotypes with cervical cytology findings. HR HPV 16 was found in 50% of HSIL reports, and “other HR HPV genotypes” were the most frequent finding for LSIL. Similarly, for women who had normal cervical cytology results, the most frequent genotypes were “other HR HPV” genotypes. According to the meta-analysis by Gary C. et al. , the mosThe burdens of HR HPV infection and cervical cytology abnormalities presented in this study are consistent with the few previous local studies and reviews in Ethiopia but somehow lower than the estimated prevalence for sub-Saharan Africa. Unlike previous studies, “other high-risk HPV” genotypes contributed considerably to the overall HR HPV burden. Multiple-type infections were found in sexually active women. The highest frequency of HR HPV positivity was in women without cervical cytology abnormalities. Hence, the interval between the primary and secondary HPV screening for HR HPV positives and negatives needs to be defined separately. The performance of the Abbott Real-Time HR HPV DNA PCR and Pap smear cytology screening methods may need to be further evaluated against histologically confirmed results. In addition, the screening program for early-age sexually active women should be further promoted in various health settings. The Ministry of Health should also further consider the possibility of introducing vaccines targeting other oncogenic HPV types in addition to genotypes 16 and 18. A large-scale community-based cohort study shall also be designed and implemented to determine the national burden and the molecular epidemiology of persistent HR HPV types and cervical cytology abnormalities which will help to recommend the ideal screening algorithm considering the local context. This will significantly contribute to the national preventive public health strategies against cervical cancer.Additional file 1:Questionnaire. (DOCX 20 kb)Additional file 2:HR HPV detection procedure using the Abbott Real-Time PCR method. (DOCX 17 kb)Additional file 3:Abnormal cytology diagnosis procedure using the conventional Pap smear method. (DOCX 17 kb)


Previous studies have shown that woman attending their first cervical screening or colposcopy appointment experience negative emotions, primarily anxiety and fear. With the introduction of the Human Papillomavirus (HPV) vaccine, it is unknown whether these emotions will have altered or whether the information needs of vaccinated women will have changed. The objective of this study is to determine the knowledge, understanding and concerns that young women have about HPV when attending colposcopy and whether their information needs are met.This is a qualitative study using semi-structured interviews which were audiotaped and transcribed. Data was analysed thematically, with recruitment until data saturation was reached. Women born after 01/09/1990 and attending colposcopy as a result of abnormal cytology were eligible to join the study. Recruitment took place in an out-patient regional colposcopy clinic, Aberdeen, Scotland.Fifteen women were interviewed. The majority of participants had some knowledge and understanding of HPV, cervical screening and colposcopy. Knowledge about the HPV vaccine was more limited; a third of participants misunderstood the effectiveness of the vaccine believing that is provided complete protection, and were left feeling that it had failed them. Some also felt that they were “test cases” for the vaccine.With the introduction of the HPV vaccine, the information and support needs of young women attending colposcopy are not fully met, leaving women with unanswered questions. With increasing numbers of vaccinated women entering the screening programme, it is timely to review the information available to these women. The development of HPV vaccination and the introduction of immunisation programmes promises the greatest impact to date on the primary prevention of cervical cancer and other HPV-related diseases . Both li. It is not known to what extent this information is read or understood.In Scotland, women born after 1/9/1990 have been offered HPV immunisation in catch-up programmes between the ages of 14 and 17 years or school-based programmes at age 11 to 13 years . Prior tIn Scotland, at the age of 20 years and 3 months, women are invited to attend for cervical screening . A natioWhilst information leaflets about screening and colposcopy have usually been developed with lay involvement, there has been limited research into the information needs of vaccinated women, and it is not clear whether their information needs are met by existing literature. As vaccinated women in Scotland are now being invited to attend cervical screening (screening starts at age 20 ), there We conducted a semi-structured interview-based qualitative study involving young women attending colposcopy.All English-speaking women born after 1/9/1990 with abnormal cervical cytology and who had been referred to colposcopy at a regional colposcopy clinic were eligible to take part. An invitation to participate in the study was sent to them prior to their clinic visit. Women were offered either a ‘face to face’ or a telephone interview after their colposcopy visit.A topic guide was developed and piloted following a literature review –13 to exInformed written consent was sought prior to interview. Interviews were audio-recorded, transcribed verbatim and anonymised. All interviews took place after participants had attended colposcopy. Participant characteristics  were obtained from screening records held in the Scottish Cervical Call Recall System (SCCRS).Analysis of transcripts started after the first participant had been interviewed and continued throughout the interview phase of the project. All transcripts were analysed thematically with themes and associated quotes logged in a Microsoft Office Spreadsheet. To ensure coding validity, three transcripts were coded separately by two independent researchers and the main coding structure was agreed upon. This coding was then applied to all further transcripts. Any additional codes were added as the analysis progressed. After 13 interviews, it appeared that no new themes were emerging; two final participants were interviewed to confirm data saturation.The study was sponsored by the University of Aberdeen. Ethical approval was granted in writing by the North of Scotland Research Ethics Committee (reference number 13/NS/0156). NHS Grampian provided R&D management approval for the study.Forty-three women were identified as eligible to join this study. Of these, 19 consented to take part and 15 were interviewed. Three women agreed to telephone interview but did not answer the telephone for the scheduled interview and one woman did not attend her face-to-face interview.Fifteen interviews were carried out between February and March 2014: eight face-to-face and seven telephone interviews. The mean length of interview was 18 min (range 15.5–22). The mean age of participants was 21.5 years (range 20–23 years). All participants were interviewed within 9 days of colposcopy with the exception of one who was interviewed 22 days later.Of the 15 participants interviewed, 13 had received the HPV vaccination and two were unvaccinated. The first unvaccinated participant had not received the vaccine as she lived abroad where the vaccine was unavailable; the second completed her secondary education in 2008 and did not take up the offer of vaccination from her General Practitioner. Two of the vaccinated participants could not recall their immunisation status and a further three could not correctly recall their number of received doses – but this information was confirmed on SCCRS for all participants.It’s [HPV] like a really, really, really common infection that most people get once in a lifetime. Many participants (10 out of 15) were aware that there are many different strains of HPV and the majority of women discussed that HPV is a sexually transmitted infection. Two participants were unable to identify the mode of transmission of a HPV infection. Participants discussed how a HPV infection was likely to resolve spontaneously without intervention but some ‘stubborn’ cases may progress into abnormal cervical cells.All participants had heard of HPV. All participants identified that the majority of women are likely to be infected at some time in their lives with a HPV infection and stated that HPV is common - Human Papillomavirus, it can create cells which can become cancerous, it’s the nature of the virus to cause cervical cancer. All participants suggested that cervical cancer was common and more likely to occur in younger women- It’s quite common in the under 50’s anyway. It’s a cancer of younger people.Of the 15 participants interviewed, 13 could identify that HPV was responsible for the change in cervical cells that may lead to cervical cancer - The vaccine is supposed to protect you against certain strains of virus. It can’t protect you against all the strains but against the main ones that cause cervical cancer. The remaining five participants discussed how they thought the vaccine provided them with complete protection against all strains of HPV – I assumed the vaccine would be 100%. I knew that it technically wouldn’t be but I thought it would be like 99.999% effective. These five participants were asked what they thought about the effectiveness of the vaccine in light of their abnormal cytology and all expressed emotions of disappointment and lack of confidence in the vaccine- The vaccine was a waste of time. I would say it’s pointless and it’s not doing what it’s supposed to. Interestingly, these five participants also expressed negative emotions regarding their colposcopy experience.Two thirds of participants (10/15) understood that the vaccine did not protect against all strains of HPV, but prevented against some types that cause cervical cancer - I don’t remember being in school and being told this [vaccine] is 94% effective or whatever. I don’t remember being told anything.When discussing HPV vaccination, one participant expressed that she could not recall receiving any information at the time of her vaccination or reading about it in an information booklet – There are other strains that cause cervical cancer. Because the vaccine only covers two of the HPV infections. Just the big ones.Considering the ten participants that acknowledged the effectiveness of the HPV vaccine, only two women understood that they were likely to be infected with a type other than 16 and 18 -My year was the first year […] Maybe it will improve in years to come. The women who expressed these ideas were amongst the oldest in the study and mentioned phrases such as ‘test group’ and ‘Guinea-pig group’.The other eight participants rationalised their abnormal test result by creating assumptions about the vaccine. Several participants suggested that they were the first group of girls to receive the vaccine, and as a result, it may not have been effective at the time -It’s different now. It’s been researched and changed. […] I hope that other girls aren’t thinking ‘Well I’m fine because I’ve got it [vaccination]’. This participant may be referring to the change in vaccine type used in the UK: In 2012 the bivalent vaccine (protecting against HPV 16 and 18) was replaced with a quadrivalent vaccine, which protects against HPV 6 and 11  [Another participant suggested the original vaccine was not effective and needed improvement. She mentioned that other young women may assume they are completely protected against HPV by the original vaccine - l warts) .I still think it’s worthwhile getting it [vaccine]. Obviously it wasn’t effective for me but I wouldn’t change it looking back.The majority of participants agreed that they would still promote the HPV vaccine to young girls regardless of their own experiences of HPV - Well obviously it’s [vaccination] not been effective for me but that’s probably because I might have had HPV before it.One participant reasonably suggested that she may have already had a HPV infection prior to her vaccination - Participants were asked whether the information leaflets they received regarding HPV vaccination, cervical screening and colposcopy were useful or reassuring, and whether they would recommend a change to the information.I don’t think any of the booklets you get for your appointments are useful. It doesn’t explain enough about what’s going on in depth. If you’re going for something like that [colposcopy], you want to know a lot more about it and understand everything about. However, none of the woman identified any specific additional information that could be included in the screening or colposcopy leaflets.Several women indicated that they read the leaflets but were still unsure what to expect during a cervical screening or colposcopy appointment: It’s very basic. It’s not very interactive. The cervical screening book is [interesting] but the colposcopy one wasn’t very interesting. Three participants felt that the information they received on HPV was lacking and they felt the need to undertake their own research- Well I did do some research on it ‘cause I didn’t know what it [HPV and abnormal cells] was. I got my information probably the worst way, from Google.In terms of improving the information leaflets, particularly the colposcopy information booklet, several women suggested that the leaflet could be made more attractive and more inviting to read- Adverts on the TV and then probably classes at school to teach more about it [HPV infection]. More than they would have before.Several women thought information on HPV and cervical screening should be introduced into school teaching and television advertisements - This study has shown that most participants have a reasonable understanding of HPV infection and cervical cancer, but more limited understanding about the effectiveness of the HPV vaccine.We believe that this qualitative study is the first to explore the information needs of women who have been vaccinated against HPV in a national catch-up programme and been referred for colposcopy because of abnormal cytology. Current information supplied in the national programme in Scotland includes information for vaccinated women but was developed prior to vaccinated women entering the programme. The results of this study may be helpful in identifying areas where current information for women may be improved.All participants were interviewed after attending colposcopy and therefore had opportunity to discuss any issues and gain information from the smear taker and/or the colposcopist. This means that study participants may be more knowledgeable or have different concerns (having attended for colposcopy) than women who either did not agree to participate in this study or who defaulted from colposcopy. Women who default may not read or understand the information they are sent and, as a result, those who default from colposcopy may have less understanding of HPV and cervical cancer than those that attend , 16. AltAt the level of screening, however, this failure to attend because of a false sense of protection from vaccination is not apparent: in Scotland, women who vaccinated as part of the catch-up campaign are more likely to attend for screening that those not vaccinated .A systematic review of girls’ and parents’ information needs regarding the HPV vaccination concluded that that there is a limited understanding of HPV and the purpose of the HPV vaccination . Our stuIn addition to the written information women receive, they will also have received verbal information from the colpsocopist. Our results suggest that HPV-vaccinated women referred to colposcopy may need different or supplementary information about vaccination and screening to target their concerns. This need for supplementary information may extend to vaccinated women invited for cervical screening and young girls awaiting vaccination , but we Several participants suggested that school teaching would be the best way to inform girls about what to expect when they attend cervical screening and the limits of the HPV vaccine. A teaching intervention lasting 5–10 min was shown to significantly increase the knowledge of HPV infection and vaccination in young men and women between the ages of 18–26, however the study did not consider how well participants retained the knowledge in the longer term . Given tOne concern expressed by a participant was that the vaccine used in the UK national vaccination programme had changed since she had received her immunisation. It is not clear whether she was aware that the reason for this was due to the tendering process for national procurement in the UK national health system  rather thttp://www.healthscotland.com/documents/24327.aspx. Updated colposcopy information leaflets are currently in progress.As more vaccinated women enter the screening programme, it is timely for cervical screening and colposcopy information leaflets to be reviewed. The updated cervical screening information leaflets currently used in Aberdeen Royal Infirmary, Scotland, can be viewed by following the weblink, For vaccinated women referred to colposcopy, explanation as to the different types of HPV, and why they may have an abnormal screening test, may lead to a more positive colposcopy experience.The information and support needs of young vaccinated women attending colposcopy are not fully met, leaving women with unanswered questions particularly around the effectiveness of the vaccine in relation to their cervical abnormalities. With increasing numbers of women entering the screening programme, it is timely to review the information available to these women.


To systematically evaluate the significance of squamous cell carcinoma antigen (SCC-Ag) in the prognosis of cervical cancer.  Literature from Pubmed, Embase, and Cochrane Library was retrieved to collect all English literature on the correlation between SCC-Ag and cervical cancer prognosis, and the quality of literature collected was assessed based on evaluation criteria. The heterogeneity, sensitivity, and specificity were detected using the StataSE12.0 software, and the correlation between SCC-Ag and cervical cancer prognosis as the effect variables was assessed using the hazard ratio (HR) and 95% confidence interval (CI). Moreover, the forest map and funnel plot were drawn. P = 0.002). The disease-free survival (DFS) was higher in low SCC-Ag expression patients than in high SCC-Ag expression patients . The progression-free survival (PFS) was inferior in patients with a high SCC-Ag expression .  A total of 17 articles meeting the inclusion criteria were selected. The high expression of SCC-Ag was significantly correlated with the poor prognosis of cervical cancer (HR = 2.22, 95% CI = 1.38 − 3.57,  SCC-Ag is an important prognostic factor for cervical cancer, and its high expression is significantly correlated with a poor prognosis of the disease. Cervical cancer is the third most common cancer in females around the world and the fourth leading cause of cancer death . CompareThe literature up to June 2018 was independently searched by two researchers in the library records. Pubmed, Embase, and Cochrane Library entries were retrieved. English index words included SCC-Ag, squamous cell carcinoma antigen, serum squamous carcinoma antigen, tumour, cancer, carcinoma, neoplasm, prognostic, prognosis, survival, carcinoma of uterine cervix, and cervical cancer.Publication inclusion criteria are as follows: (1) studies based on cervical cancer patients; (2) published epidemiological studies, such as case-control studies and line-up studies; (3) studies evaluating the diagnostic value of tumour markers for cervical cancer; (4) positive values or HR and 95% CI of the case group and the control group were clearly reported, or the corresponding four-table data were provided; (5) research methods and scope were similar, and detection methods were the same; (6) the relationship between SCC-Ag expression and prognosis of cervical cancer was reported; and (7) the data and information were complete.P value; (7) inconsistent publications; and (8) publications that lacked the results or controversial publications.Publication exclusion criteria are as follows: (1) publications containing the pathological type of noncervical cancer; (2) cases that had no clear diagnostic criteria; (3) reviews or conference minutes; (4) the number of cases was less than 10; (5) publications that did not provide credible case and control sources; (6) lack of raw data to calculate the HR, 95% CI, and Two researchers independently screened the literature according to the inclusion criteria. If there were differences or disputes in the process, they reached an agreement through discussion and study. The first step was to exclude irrelevant articles from the topic and abstract reading; the second step was to exclude publications without reference values; the third step was to exclude articles with no integrity and accuracy data; and, finally, the articles meeting the inclusion and exclusion criteria after screening were obtained.Two researchers independently screened the literature, extracted and cross-checked the data, searched the relevant publications according to the search strategy, screened the appropriate publications by looking through their titles and abstracts, and read the full text to determine whether the inclusion criteria were met. The information to be extracted mainly included the first author's name and nationality, publication period, number of patients, pathological stage, age, follow-up time, test samples, test methods, cut-off values, analysis methods of the survival rate, and survival analysis.I2 and P. If I2 > 50% and P < 0.05, the heterogeneity among studies was strong and the data were combined by a random effects model; if I2 < 50% and P > 0.05, there was no heterogeneity among studies and the data were combined using a fixed effects model. The Stata12.0 software was also used to draw the funnel plot to find potential publication bias.The correlation between SCC-Ag expression and cervical cancer prognosis as the effect variables was evaluated by the HR value and 95% CI. If the HR and 95% CI were described in the publication, they were extracted directly. If not described, they were calculated based on available data. Data for the Kaplan-Meier survival curve were obtained by the Engauge software. A meta-analytical method was used to calculate and merge the results of the publications that met the inclusion criteria, and the Stata12.0 software was used to draw the forest map. If HR > 1, patients with a high SCC-Ag expression had a worse prognosis; if HR < 1, patients with a high SCC-Ag expression had a better prognosis; and if HR = 1, the high expression of SCC-Ag was not related to the prognosis of patients. The heterogeneity of this study was investigated by A total of 387 publications related to SCC-Ag expression and the prognosis of cervical cancer were retrieved. Of these, 348 publications unrelated to the study were excluded by carefully reading the title and abstract, and the remaining 39 publications were further examined by screening the full text. Finally, 17 publications were studied, including 4 that simultaneously analyzed overall survival (OS) and progression-free survival (PFS) as prognostic indicators, 4 that analyzed OS and disease-free survival (DFS) as prognostic indicators, 5 studied with DFS alone, 3 articles with the most endpoints of OS alone, and 1 studied with PFS alone  6–22]. –22. 6–22I2 = 91.4%, P < 0.001). The random effects model was used to combine the HRs. The pooled results showed that the high expression of SCC-Ag was significantly associated with the poor prognosis of cervical cancer . The forest graph is shown in I2 = 23.3%, P = 0.236), and the combined effects model was used to merge the HRs  , and HR was combined using a random effects model   . There w= 0.028) . The cauP = 0.038) and those for the HR-extrapolated group were HR = 3.92 . In the racial analysis, the results for East Asian were HR = 1.99  and those for Caucasian race were HR = 2.78 . Multivariate analysis of prognostic DFS subgroups resulted in HR = 2.23 , whereas the univariate analysis results were HR = 2.05 ; the results for East Asian were HR = 1.95 , and those for Caucasian race were HR = 2.57 .According to the clinical characteristics and the results of the meta-analysis, the studies providing OS  and DFS The publication bias was determined by Egger's test and Begg's test, and the resulting funnel plot was asymmetrical, as shown in α in diagnosis [SCC-Ag is a glycoprotein, a subtype of the tumour-associated antigen TA-4, with a molecular weight of approximately 45 kDa. It is a clinically important tumour marker. Kato and Torigoe invented a radioimmunoassay for simple and convenient detection . It has iagnosis . Cervicaiagnosis , 28. In iagnosis . Serum SP = 0.001). However, the cut-off values used in these studies were all different. The most used cut-off value was 1.5 ng/mL, which may be a criterion for predicting prognosis, but it needs to be further verified, and there may be errors in some data extraction processes. In addition, the follow-up time of each study was different, and some patients were lost to follow-up or were false positives. Moreover, the literature included a retrospective study that may affect the accuracy and authenticity of the data. Therefore, the reasons for the obvious heterogeneity of this meta-analysis may be the inclusion of English literature, the small number of included publications, tumour stage, cut-off value, follow-up time, and other factors. Finally, although this is the only marker assessed in the study, we should realize its limitations that other factors may be more sensitive. Future research needs to focus on the combination between SCC and other important factors of cervical cancer. In the clinical treatment of cervical cancer, it is necessary to accurately diagnose the patient, analyze the patient's pathological stage and lymphatic metastasis, dynamically observe the changes in the patient's SCC-Ag, and implement remedial measures in advance to improve the patient's quality of life and reduce their pain.In the meta-analysis, it was found that the high expression of SCC-Ag was significantly associated with the poor OS of patients with cervical cancer (HR = 2.73, 95% CI = 1.48 − 5.05, This meta-analysis summarizes all studies of SCC-Ag and cervical cancer prognosis, indicating that the high expression of SCC-Ag is significantly correlated with poor OS in cervical cancer. Considering the limitations of the study, further prospective and large-sample studies are needed to confirm the prognostic value of SCC-Ag in cervical cancer and to provide evidence for a better investigation of treatment strategies.


To evaluate the treatment effect of genital warts, we investigated the quadrivalent HPV vaccine injection compared with surgical excision.This prospective study included 26 patients (M:F = 24:2) who received HPV vaccine or surgical excision. After explanation of surgical excision or HPV vaccine, 16 patients underwent surgical excision and the others received HPV vaccine injections. Based on gross findings of genital warts, treatment outcomes were classified as complete response (no wart), partial response, and failed treatment.Among enrolled patients, 42% (11 / 26) patients had recurrent genital warts. In vaccination group, complete response rates of genital wart were 60% following 3 times HPV vaccine. Partial response patients wanted to excise the genital lesions before the 3 times injection, because they worried about sexual transmission of disease to their sexual partners. One patient underwent surgical excision after 3 times injection. Excision sites included suprapubic lesions, but other sites including mid-urethra and glans showed complete response after injection. At a mean follow-up period of 8.42 ± 3.27 months, 10 patients (100%) who received HPV vaccine did not show recurrence.The response rates after HPV vaccine injection were 90% . Our results suggested that HPV vaccines could be effective in management of genital warts. Human papillomavirus (HPV) is the source of the most common sexual transmitted disease, which could infect the mucosa and skin of the anogenital regions. Infection with HPV causes a large proportion of uterine cervix, vaginal, vulvar, anal, and penile cancers, as well as genital warts. Completion of the HPV vaccine series is important to protect adolescents against the most common HPV types associated with cervical and penile cancers as well as genital warts before they are exposed to the virus . High riTherefore, the Advisory Committee on Immunization Practices recommends HPV vaccination for girls and boys aged 11 or 12 years. Routine recommendation is quadrivalent HPV vaccine for girls and boys, or bivalent vaccine for girls at the age of 11 or 12 years . QuadrivLow risk HPV types, such as type 6 and 11 are the most frequent cause of genital warts . They arFrom January 2015 to December 2016, a prospective trial was conducted on 24 male patients and 2 female patients who received HPV vaccine or underwent surgical excision. Patients were evaluated for medical history, and physical examination was conducted by routine laboratory studies before vaccination. If urethral involvement was suspected, cystourethroscopy was performed.Recurrence of HPV was also inspected by noting the history and included 14 patients who were treatment naive, while the remaining patients had received repeated freezing, laser therapy, electrocautery, and / or other treatments.Numerous HPV lesions within clear boundary masses with mostly wide pedicle and moist smooth surface were observed. Occasional fused lesions were also present in some patients and the majority of lesions were gray-brown in color, while a few were dark red. The HPV lesions were counted carefully. All patients were confirmed to have condyloma acuminatum by pathological review.The potent therapeutic effect and an additional preventive effect of the HPV vaccination treatment was discussed with the patients. Then, the patients decided if they should be vaccinated initially and undergo definite treatment later if there was no response after vaccination. In cases of the patients who underwent definite surgery first, we reviewed the reason by a survey.The HPV vaccine injection group received 3 doses  within 6 months. After first injection, the second injection was done around 2 months later and the third injection was administered around 6 months after first injection.Surgical excision of the condyloma was performed using a surgical blade. At first, masses were excised as much as possible by blade then electrocauterization was done optionally in bleeding focus.Treatment outcomes were assessed based on the gross findings of the condyloma lesion. After vaccine injection or surgical excision, no gross condyloma lesion was classified as complete response. The cases in which more than half of the lesions disappeared by treatment, were defined as partial response. In addition, if more than half of the lesions remained or aggravated at the follow-up visit after third injections in 6 months, the patients were classified as failed treatment.We conducted a prospective trial using 26 cases of HPV genital infection. About 42% of the patients had a history of genital warts that recurred more than once. Absolute results of recurrent infection were 60% in vaccine injection patients and 31.25% in surgical excision patients. In case of the HPV lesion, only one condyloma was counted in 18.75% / 30%, two to four in 18.75% / 30%, more than five in 62.50% / 40% in surgical excision and vaccine injection patients, respectively.Complete response rates of genital wart were observed to be 60% following 3 HPV vaccine injections at a mean follow-up period of 8.42 ± 3.27 months. In a case of complete response by vaccination, a 36-year-old female patient had multiple HPV lesions on the external genitalia and around the urethral meatus as shown in Among 16 patients who underwent surgical excision, 8 (43.75%) had expensiveness as the major concern, followed by 3 who were worried about treatment failure (18.75%), 2 worried about sexual transmission (12.5%), 2 worried about side effects (12.5%), and one patient did not answer the question.HPV is a DNA virus from the papillomavirus group with more than 170 family types . HPV resMost of HPV infections are temporary and asymptomatic and raise no medical problems. Seventy percent of new HPV infections are self-limited within 1 year, and almost 90% are cleared within 2 years.The duration of new infections is 8 months in average . The risIrrespective of the localization, HPV invades until the glandular epithelium of the cervix resulting in glandular cancers, such as carcinoma in situ or invasive adenocarcinoma . The HPVThe natural history of naturally acquired immunity related to HPV is poorly understood. HPV vaccine has been valid for routine vaccination of juvenile girls since 2006. It is true that HPV vaccination has been mainly focused on young female patients. Irrespectively, HPV vaccine has been proven to also be useful in male patients for the prevention of HPV recurrence and its associated malignancies. Recently, HPV vaccine has been permitted to be used for male patients in several countries as well , 18. TheConsequently, HPV vaccine has been shown to be potent in preventing genital warts and related cancers in both genders. Vaccine is a medical agent that generates antibody and provokes a subclinical immunological effect in the human body by enhancing immunity to the specific infection. HPV creates warts in various proportions of the cervix, vagina, and vulva in female patients; the penile and scrotum areas in male patients; as well as the anal area in both genders. Therefore, completion of the HPV vaccination series is essential to protect patients against the most common HPV types associated with genital warts previous to the exposure of HPV.Recently, the idea of vaccination was extended to the concept of its therapeutic usage. Sustained expression of the HPV oncoproteins, E6 and E7 are crucial factors in the development of intraepithelial neoplasia and cancers, and thus, creating main targets for therapeutic vaccines .In the cervical HPV infection, complete response of cutaneous warts was noted after 3 injections of the vaccine . AnotherEven though HPV vaccine was intended to produce neutralizing antibodies to avoid reinfection, its abilities to induce cell-mediated immunological responses were confirmed in previous studies , 23. AddSome previous clinical trials assessing the outcomes of therapeutic HPV vaccinations showed unsatisfactory results, and studies on therapeutic vaccines against HPV are mainly being published as case reports as discussed above . On the Recent experimental study on the combined use of the imiquimod 5% cream and quadrivalent recombinant HPV vaccination to achieve long-term clinical remission of chronic HPV infection manifested by anogenital warts was published. This study enrolled 36 young patients (22 men) that were vaccinated by quadrivalent recombinant and co-administered with imiquimod 5% cream three times in a week up to four months. Complete remission of genital warts was observed in 94.4% patients within 1 year and 2 patients with anogenital warts were treated with Solcoderm® cream which completed the removal of genital warts. There were no recurrences throughout follow-up period .Another report showed noninferiority of the immune reaction in males (age 10-15 years) compared with same aged female group, and in males (age 9-15 years) compared with same females group it was demonstrated that both geometric mean titer and seroconversion rates for all four HPV vaccine types met non-inferiority criteria in the per-protocol population. So we may expect the similar treatment effect irrespective of gender .Our case series is the first to reveal the therapeutic effect of HPV vaccination. The response rates of 90%  after vaccine injection were promising. Besides, our idea of therapeutic vaccination on the genital warts is based on some valuable merits. Firstly, genital warts are not a fatal disease, so we can choose a treatment from multiple options considering various factors, and definite surgical treatment could be deferred after systemic vaccinations. Additionally, therapeutic vaccination is relatively non-invasive, because surgical management may induce pain and scar formation. Furthermore, we should be cautious about viral seeding during ablation procedure. If HPV infects the cervix, approximately 90% of the infections are cleared without any management within 2-3 years even though variation depends on their type and risk of HPV. Cellular immunity plays an important role in this natural loss of HPV infection , 30. ThiHPV vaccines as a treatment option have to overcome several hurdles. Therapeutic vaccination has not been officially evaluated in a randomized prospective trial and accordingly it has not received a formal approval in any country. Therefore, there remains a critical necessity for clinical trials to evaluate the efficacy of HPV vaccination as a treatment. We need to understand the difference in clinical response of patients after vaccination for treatment of HPV infection. Various concerns of patients who underwent curative resection, such as expensive cost, treatment failure, sexual transmission, should be overcome by cost analysis or education about the barriers during the treatment period.Some patients who were administered with HPV vaccine for the management of genital warts, were worried about sexual transmission to their partners. They underwent surgical excision during the vaccine administration period and the infection did not recur during the follow-up. So, to maximize the efficiency of the vaccine, the possible candidates should be vaccinated irrespective of the gender and condition of the patients.This research has several limitations, such as small number of patient group and no prospective randomized control group. Additionally, we do not have HPV typing result and biomarkers for immunological responses or risk level. We explained potent therapeutic effect of vaccine that was not fully proven for genital warts. So this may introduce a very strong bias and misinformation in the consent process. Patients were mixed with the first episode and recurrent episode, so the immunology issues may be different in both cases. Furthermore, we do not know the optimal dose or regimen enough to protect and we should not know the age-related treatment option even many countries are not limiting the age for people to get the prophylactic vaccine.Even though our study has some drawbacks, our data on therapeutic vaccination shows its potential benefit as an excellent treatment candidate to motivate clinical regression in genital warts.Genital warts remain a serious issue worldwide because early diagnosis is difﬁcult and proper primary management is sometimes missing. In the absence of vaccine coverage, the presence of high number of HPV infections, and the incidence of HPV-related tumors will remain a global public concern worldwide, including Korea. In conclusion, classical concept of vaccination is to generate beneficial immunological effect in the body to prevent recurrences. In our opinion, therapeutic HPV vaccination can eliminate genital warts, leaving people with better protection against future recurrences caused by HPV relapse.The response rates of our research after therapeutic HPV vaccination indicated effectiveness as an initial management approach of genital warts with potent preventive advantages over recurrent infection.


Human papillomavirus (HPV) infection may lead to a series of lesions in the cervix. Distributions of HPV genotypes reveal that an increased prevalence of high-risk HPV (HR-HPV) is positively correlated with the severity of cervical lesions. Furthermore, persistent infection of HR-HPV is associated with a risk of cervical cancer. Considering the newly approval of the HPV vaccine in China and the prevalence of HPV distribution, which is meaningful for directing efforts for HPV vaccination, a more detailed understanding of HPV distribution is critical. This study aimed to investigate the overall prevalence of HPV and the age-specific features related to HPV distribution in the Jiangsu population.We collected a total of 62,317 cervical cytological specimens from Xuzhou, Nanjing and Suzhou, which represent the northern, middle and southern regions of Jiangsu Province, respectively. All these samples were assigned to 6 groups based on participant age. HPV genotypes tests were performed by using a commercial kit which is designed for the detection of 17 high-risk HPV genotypes and 6 low-risk HPV genotypes.The overall prevalence of HPV was up to 26.92% in Jiangsu Province. The most common high-risk genotype was HPV52 (5.09%), followed by HPV16, HPV58, HPV53, HPV51 and HPV68. The most prevalent low-risk genotype was HPV81 (2.70%), followed by HPV43, HPV42, HPV6, HPV11 and HPV83. Most infections were caused by HR-HPV, while single-genotype infection occurred more frequently than multiple-genotype infection. Regarding participant age, the overall infection rate of HPV was distributed in a U-shaped manner, with the highest peak in the younger than 20-year-old cohort. Additionally, significant variations were found between different cities, representing different regions of Jiangsu.HPV prevalence is high in Jiangsu Province. The prevention of HPV-related diseases is challenging. Given the variation in HPV prevalence between ages groups and regions, a flexible HPV vaccination program, adjusted base on regional infection features, could have a beneficial effect in Jiangsu Province. The strong correlation between cervical HPV infection and cervical lesions or cervical cancer has been well established. HPV infection is the most common sexually transmitted infection. More than 200 HPV genotypes have been identified, with approximately 40 distinct HPV genotypes affecting the mucosal epithelium of the genital tract. HPV genotypes are typically divided into two groups according to their carcinogenicity: the high-risk group (HR-HPV) and the low-risk group (LR-HPV) . DistribFollowing the release of the Gardasil 4 HPV vaccine in mainland China, prevention of cervical cancer became widely discussed among the public. HPV vaccine administration in subjects between 26 and 45 years of age has shown safe, effective protection against vaccine-related genital warts and cervical dysplasia . The preFrom April 2013 to August 2017, 62,883 cervical samples were collected from women attending routine outpatient gynecological examinations in three cities in Jiangsu Province. For this study, the total of 62,317 cervical samples were acquired from the three included cities regions, and the overall infection rate along with the type-specific infection rate was calculated. The prevalence of HPV infection among different regions and different age groups was compared. Participants were enrolled based on the following criteria: no sexual activity in the previous 48 h and no use of vaginal medication or washing in the previous 48 h. Women who were menstruating or pregnant were excluded.The study population consisted of gynecological outpatients and asymptomatic women. They were devided into 6 age groups in the study.All the samples and their clinical information were obtained following protocols approved by the ethics committees of the local hospitals, and the written informed consent was signed by all the participants.Cervical cell scrapings were collected with a cytobrush included in the testing kit provided by the company. The cervical samples were kept in a sample transport medium for HPV detection and a cell preservation solution for cytology testing. All samples were shipped to the lab at 4 °C and the test was performed within 48 h. The specimens were kept at room temperature for no longer than 12 h before examination. Specific HPV genotypes were using the HPV Genoarray test kit, a PCR-based hybridization gene chip assay.HPV detection and genotyping were performed using a commercial HPV Genotyping Kit for 23 HPV types , including 17 HR-HPV types  and 6 LR-HPV types . This kit has been approved by the China Food and Drug Administration (CFDA). The protocol, including DNA extraction, polymerase chain reaction (PCR) and reverse dot blot hybridization (RDB), are described specifically below. All the experimental procedures were strictly followed the manufacturer’s instructions. DNA extraction, PCR amplification and genotypes detection were conducted as follows.Firstly, the liquid-based cytology specimen was centrifuged in an Eppendorf tube. After the supernatant was discarded, DNA was extracted. DNA concentration and purity (OD260/OD280 between 1.6–1.8) were examined using a spectrophotometer to determine. Next, PCR was carried out. Amplification was started with initial denaturation at 95 °C for 5 min, followed by 5 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and elongation at 72 °C for 30 s. An additional 35 cycles were conducted, carried out including denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s and a final extension at 72 °C for 3 min. The kit employs DNA amplification to detect HPV positivity in a microarray format, utilizing a nylon membrane onto which HPV genotype-specific oligonucleotide probes secured have been immobilized to simultaneously identify 23 HPV genotypes. The blue spots on the strip test strip, indicating a positive result, could be discerned by the naked eye.The screened population was divided into 6 groups based on their ages. The T1 group indicates age ≤ 20; the T2 group indicates 20 < age ≤ 30; the T3 group indicates 30 < age ≤ 40; the T4 group indicates 40 < age ≤ 50; the T5 group indicates 50 < age ≤ 60; and the T6 group indicates age > 60.2 test was utilized to evaluate the significance of difference between designated groups. All tests were two-sided and interpreted as being significant at p ≤ 0.05.All statistical analyses were performed by using SPSS 19.0 statistical package for Windows. A Xp < 0.05), with a lower prevalence in Xuzhou, and a higher similar prevalence rates in Nanjing and Suzhou. The six most prevalent HR-HPV HPV genotypes in Xuzhou and Nanjing were 52, 16, 58, 53, 51, and 68; however the top six HR-HPV genotypes in Suzhou were 16, 58, 52, 33, 18, and 68. The two most prevalent LR-HPV genotypes in Xuzhou and Nanjing were 81 and 43, but in Suzhou the most two common LR-HPV genotypes were 11 and 6  of HR-HPV infection occurred in Suzhou Table . Age-speIn the present study, the presence of HPV infection among the females who underwent the gynecological examination in three cities of Jiangsu Province was tested utilizing a commercially available HPV DNA kit. The overall HPV prevalence in Jiangsu province is 26.92%, which is a higher rate than was found in a previous study in Zhejiang (20.54%) , and lowNotably, the prevalence of HPV infection was significantly different between age groups . The firFrom the data we collected, single genotype infections were observed more frequently than multiple genotype infections, and made up 54% of the total infections. The most common type of multiple infection was a double infection. HPV16 and HPV52 were the most frequent combination in Xuzhou and Nanjing, but in Suzhou, the most frequent combination was HPV16 and HPV33. The maximum quantity of different genotypes in a multiple infection was 9 HPV genotypes. It has been reported that multiple infections often promote the development of cervical diseases , increasIn conclusion, we performed a population-based study aimed at detecting HPV prevalence in three representative cities of Jiangsu Province. The age-specific and region-specific infection rates demonstrated significant variance; this finding will provide guidance and increase the accuracy of future vaccination programming. Further study should focus on infection patterns, as demonstrated by the correlation between pathology and infection genotypes. It is important to discover which populations are inclined to persistent versus transient infections, as this understanding is crucial for precise vaccination of the target population.


The differences between high risk and low risk human papillomaviruses  that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6, as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that, as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV. There are currently 198 HPV types that have been officially numbered4, of which approximately a third are predominantly detected in the cervical epithelium and belong to theAlphapapillomavirus genus6. The viruses of this genus are further grouped into high-risk (HR) and low-risk (LR) HPV types based on their association with cervical cancer and pre-cancerous lesions8. Most of the HR-HPV variants belong to theHuman papillomavirus 16  orHuman papillomavirus 18  species groups9.Persistent infections with carcinogenic human papillomaviruses (HPVs) are the main cause of cervical neoplasia and cancer, with over 99% of the cervical lesions containing HPV sequences11. The early HPV proteins E6 and E7 have transforming properties14 and are required for the malignant conversion. The involvement of these proteins in tumorigenesis is thought to stem from their interactions with the tumor suppressors p53 and pRB, respectively, as well as other proteins involved in tumorigenesis20. Variations in E6 and E7 proteins have been suggested to determine the oncogenic potential of HPV22 but the potential discriminating features of the oncogenic variants are frequently observed in LR-HPVs as well27. A notable molecular feature that distinguishes HR from LR-HPV  is the presence of a PDZ-domain recognition motif at the extreme C terminus of the HR-E6 oncoprotein, as opposed to LR-E630, which enables interactions of HR-E6 with numerous PDZ domain proteins33.Phylogenetic trees constructed from alignments of complete HPV genomes cluster all oncogenic types together, suggesting a common ancestor for the HR-HPVs. However, in separate trees built from different regions of the genome, the carcinogenic potential co-segregates with the early (those produced prior to replication) but not with the late genes35. Alternative splicing of introns in the E6 gene produces multiple splice isoforms of E6-E7 pre-mRNA34,36. In HR-HPV16 and HPV18, E6*I is one of the major splice isoforms that functions as the mRNA for the production of E7 via translation reinitiation37, In contrast, unspliced E6 mRNA is responsible for the production of E637. Another recent study has shown that HPVs generate single-stranded circular RNAs (circRNAs), some of which encompass the E7 gene (circE7)38.Both oncogenes are transcribed from an early promoter as a single E6-E7 polycystronic pre-mRNA. The transcriptional level and translational efficacy of this RNA are regulated by the alternative RNA splicing machinery of host cellsIdentification of signatures of the HR-HPV genotypes that differentiate them from the majority of alpha papillomaviruses that lack oncogenic potential could help elucidate the genetic basis of the carcinogenic properties of HPVs, thus contributing to a better understanding of the biological mechanisms exploited by the virus to trigger neoplasia. However, at present, genomic determinants of the HPV oncogenic risk are not well understood, and the exact nature of the genetic changes that led to the emergence of the HR-HPV oncogenicity remains unknown.42. We hypothesize that the insert separating the coding sequence of E6 and E7 was the primary cause of the emergence of high oncogenic potential alpha-HPV.To better understand HPV carcinogenesis, we revisited the search for specific genomic determinants of HR-HPV types and identified a previously unreported insert of 30 to 60 base pairs (bp) at the 3’-end of the E6 oncoprotein coding region that is present in all HR-HPV types but not in LR-HPV. This insert introduces a new stop codon, separating the nucleotide sequence coding for E6 from that coding for E7, thus, eliminating the overlap between E6 and E7 that is characteristic of the LR-HPV types. The insert confers a PDZ binding motif at the end of E6 oncoprotein which is likely important for the oncogenic potentiation. Additionally, the insert places the termination codon of E6 upstream but in close proximity of the initiation codon of E7. Furthermore, the insert contains sequences complementary to human 18S rRNA in the regions of hairpins 26 and 27 that are known to interact with viral RNAs and are specifically involved in IRES  binding and cap-independent translationpave.niaid.nih.gov43). We then constructed a global multiple sequence alignment of the whole genome nucleotide sequences and the amino acid sequences alignments for each protein. Maximum likelihood phylogenetic analysis of these alpha-HPVs, based on the complete nucleotide sequence, as well as the amino acid sequences of most of the early proteins, identified HR-HPV as a clade, whereas phylogenies of L1 and L2 did not support the monophyly of the HR-HPV . We then applied Support Vector Machine (SVM), a linear classification technique, with a leave-one-out cross-validation, aiming to identify regions that differentiate between high-risk and low-risk strains. This approach resulted in the identification of genomic regions that separated HR-HPV from LR-HPV with high accuracy . Among these, we found one prominent insert (between 30 and 60 nucleotides) located in the 3’-terminal region of the E6 gene (44 matrix (seeMethods). As expected, the divergent region in E6 was identified from the amino acid sequences as well .First, we searched for regions of insertions and deletions within the genome nucleotide sequences of E6 and E7 that might differentiate between the risk groups. Specifically, we identified sequences with high frequency of deletions or insertions that are located within high confidence alignment regions . Combined with the separation between the coding regions of E6 and E7 resulting from the insertion, and the proximity of E6 stop codon to the E7 start codon, this finding led us to hypothesize that the insert has an additional role as a regulatory element. Furthermore, as E7 has been previously identified as the dominant oncogene50, the lack of genomic determinants of HR-HPV within the E7 gene is compatible with the possibility that the insert contains regulatory elements enhancing E7 expression in HR-HPV strains.We observed that the sequence similarity between the insert sequences among HR-HPV strains is more pronounced at the nucleotide level than at the amino acid level . The second region of 18S RNA complementarity overlaps with the E7 start site, and hence might function independently or cooperate in the regulation of E7 translation , some of which encompass the E7 gene (circE7)Alphapapillomavirus includes HPV types that are uniquely tumorigenic. However, the events in the HPV genome evolution that led to the carcinogenic potential of some alpha-HPV types remain poorly understood. Here, we revisited this problem by performing a search for genomic determinants of the oncogenic risk of alpha-HPV types and identified a unique insert in the 3’-terminal regions of the E6 oncoprotein genes in all HR-HPV strains. To the best of our knowledge, this insert in HR-HPV genomes has not been reported previously. The insertion maintains closely similar lengths of E6 proteins in HR-HPV and LR-HPV types, which could explain why it has been overlooked in previous HPV genome analyses.The genus61. Interestingly, PDZ-binding motifs have been identified also in several other oncogenic viruses, such as HTLV-1, adenovirus RhPV1 and beta-HPV863. Second, the insert eliminates the overlap between the E6 and E7 coding regions, implying a possible role as a regulatory element. We found that almost all HR-HPV genomes contain the sequence T-A/G-T-A-A-T/A in the insert near the end of the E6 coding sequence, which is closely similar to the sequence of the early promoter at the 5’ end of E6 that is employed for the synthesis of the E6-E7 mRNA65. However, for the HR-HPV strains, unlike the case of the LR-HPV66, there are no reports of an independent E7 promoter, so that E6 and E7 are both translated from a polycistronic mRNA. Thus, the promoter-like sequence within the insert is likely to be spurious.We hypothesize that the insertion makes a dual contribution to the oncogenicity of the HR-HPV types. First, the inserted sequence changes the C-terminal amino acid sequence of E6 and creates a PDZ domain-binding motif that is unique to HR-HPV types. The experimentally demonstrated interaction between the E6 proteins of HR-HPV and several PDZ domain-containing proteins is thought to contribute to HPV-induced tumorigenesis67, and translation reinitiation has been suggested as the mechanism68. However, the close proximity of the E6 stop codon to the E7 start codon in HR-HPV (only a few nucleotides separating these codons) could inhibit re-initiation68. Therefore, it has been suggested that ribosomal reinitiation is enabled through the formation of the E6*I splice variant  in which the intercistronic distance between the translation termination codon of E6*I and the E7 initiation codon is increased37. Thus, E7 protein translation might be enhanced by the removal of the intron I region from pre-mRNAs by splicing, whereas retention of that sequence is required for E6 translation69. A comprehensive characterization of HPV16 and HPV18 expression by RNA-Seq analysis in invasive cervical cancer has shown that E6*I is the most abundant transcript isoform for both viral types70. The insert identified here is located in the 5’UTR immediately upstream of the E7 start codon in the E6*I transcript, and there are two alternative branch sites for E6*I splicing to produce a E7 mRNA with variable 5’UTRs71. However, several studies have reported that E7 translation is independent of splicing within the E6 open reading frame73 , undermining this interpretation.In HR-HPV strains, E6 and E7 proteins are translated from a polycistronic pre-mRNA59. The first motif forms an internal loop in the predicted mRNA secondary structure of E6-E7, whereas the second one overlaps with the E7 initiation codon. Given the evolutionary conservation of these motifs and the close proximity of E6 termination site to the E7 initiation site, it appears plausible that coupled termination-reinitiation promoted by the insert sequence is an important mechanism for E7 translation in HR-HPV strains.Further investigating the potential regulatory role of the inserted sequence, we identified two conserved motifs that are complementary to the human 18S rRNA ; interacE6E7 pre-mRNAs, but also in some alternatively splicedE6E7 transcripts, including the most common spliced isoformE6*I in HR- HPV16 and HPV1870. The biologically functional circE7 that has been identified in TCGA RNA-Seq data from HPV-positive cancers38, contains the unique insert in its 5’UTR. Thus, the regions of complementarity to human 18S rRNA could modulate the translation rate of the E7 encoded by alternative transcripts and circRNAs in HR-HPV.The folding of these regions of rRNA complementarity in E6-E7 mRNAs is typically relaxed in the predicted optimal and sub-optimal secondary structures of the HR-HPV strains. The insert is present not only in polycistronic74, in which the E6 gene was found to be highly variable whereas the E7 gene was strikingly devoid of genetic variants in precancer and cancer cases, support the notion that idea that E7 is a the contributor in HR-HPV-induced carcinogenesis75. Thus, enhancement of E7 production by regulatory elements contained in the insert is likely to substantially affect the oncogenicity of the HR-HPV strains76. The insert is likely to enable coupled termination-reinitiation of the E6 and E7 genes, which is supported by the presence, within the insert sequence, of motifs complementary to the human 18S rRNA. In addition to the enhancement of reinitiation during the E6-E7 mRNA translation, this interaction might affect the dynamics and efficiency of E7 translation from alternative transcripts in HR-HPV. Furthermore, these motifs might also regulate E7 translation encoded by circE7 through cap-independent mechanisms. The dynamics and the rate of translation of E7 alternative isoforms appear to vary and seem to influence the stability of E7 proteins differentially across HPV types76. Thus, potential 18S rRNA – insert duplexes could modulate the rate of E7 translation initiation and elongation in HR-HPV and result in differences in stability of HR-HPV E7 proteins compared to LR-HPV, and thus could affect the interactions between E7 and various protein partners.Recent analysis of 5570 cervical HPV16 genomesGiven the lack of additional major genomic determinants consistently differentiating between HR-HPV and LR-HPV, it seems most likely that the insert in the E6-E7 transcript is the primary cause behind the emergence of oncogenic HPV and makes a complex contribution to the oncogenicity of the HR-HPV types in which both E6 and E7 oncoproteins are involved.43) were generated usingMAFFT v7.40777 with default parameters. Maximum likelihood phylogenetic analysis was performed using the resulting alignments andPhyML 3.1 software78, with the Bayesian information criterion, NNI tree improvement and an LRT SH-like likelihood method for support estimation.Multiple alignments of nucleotide and amino acid sequences that were obtained from PaVE  to the nucleotide sequences, we first encoded the data with numbers, where each nucleotide is coded as ‘1’ and each gap as ‘0’. We searched for alignment regions with deletions or insertions in multiple HPV strains that are surrounded by high confidence alignment regions  because these are most likely to contain relevant differences within conserved genomic regions. Within these regions, we then trained the SVM to classify alpha-HPV strains based on their oncogenic potential. The performance of the SVM was evaluated by leave-one-out cross validation, and regions with the overall balanced accuracy >0.75 (the average of the accuracy for positive and negative classes) were selected for further analysis.To apply Support Vector Machines (SVMfitsvm function) to the amino acid sequences of E6 and E7 proteins, we first encoded the amino acids with numbers using the frequencies of each amino acid in each protein and the BLOSUM62 matrix44. For each position, the most frequent amino acid was identified, and the amino acids in each protein were encoded by their BLOSUM62 distances from the most frequent amino acid in the respective position. We then trained the SVM to classify alpha-HPV strains based on their oncogenic potential using the coded protein sequences. Positions surrounded by high-confidence alignment regions (length > 5 amino acids and containing less than 5% of gaps in each position) were selected for further analysis. For these positions, we evaluated the performance by leave-one-out cross validation, and regions with the overall balanced accuracy >0.75 were selected for further estimation of significance using a permutation test.To apply SVM (using Matlab 2018bExtended data62), controlling for the topology of the reconstructed phylogenetic trees. To this end, the labels were randomly permuted while maintaining unified labels for clades with high similarity. For each identified region, the likelihood of obtaining an equivalent or higher performance for the length of the region within the respective protein was evaluated by calculating an empirical P-value. We consider regions with permutation P-value <0.05.To estimate the significance of the identified regions, i.e. to determine whether similar differences could be observed by chance, we performed a permutation test  obtained from a leave-one-out cross validation for predicting risk category (HR vs LR) of alpha-HPV strains using BLOSUM62 coding of amino acid sequences, of different positions (x-axis) E6 (A) and E7 (B). Zero-accuracy was assigned to regions surrounded with low confidence alignment.• Figure S3: Boxplots showing the distributions of the identity fraction of each nucleotide (NN) and amino acid (AA) in the genome and protein sequences of the insertion (not considering gaps for both NN and AA). The individual identity fractions of each position are overlaid.• Figure S4: RNA fold secondary structure prediction of HR HPV strains 16 (A), 18 (B), 45 (C) and 31 (D). The nucleotides of the first motif are marked in red, and of the second motif in purple. E7 AUG is noted in red font.Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of thehttp://doi.org/10.5281/zenodo.324223185. Apache License, Version 2.0.Zenodo: Permutation test controlling for HPV strains, I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I approve the manuscript in its revised form.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this study, Auslander and colleagues compare nucleotide and amino acid sequences for a subset of genus alpha HPV genotypes. They observe that high-risk HPV genomes share a short sequence insert at the 3’ end of the E6 ORF. The insertion adds a PDZ binding motif to the C-terminus of high-risk HPV E6 proteins and alters the location of the E6 termination codon relative to the E7 initiation codon. This observation is consistent with previous findings. It has been appreciated for some time that high-risk HPV E6 and E7 are transcribed from a polycistronic mRNA whereas low-risk E6 and E7 are transcribed from separate promoters. In addition, a frequent splicing event occurs within the E6 gene in the bicistronic high-risk HPV early mRNA. This report adds to those observations. It proposes that the 3’ high-risk specific insert is another feature that differs between high- and low-risk HPV and that it might drive differences in expression of high-risk versus low-risk HPV oncoproteins. Understanding the differences between oncogenic and non-oncogenic HPV is an area of intensive research and new contributions in this area are potentially significant. This report makes a useful contribution to the literature.1; others were identified by proteomic analyses from several groups23. For example, the authors do not mention that TP53 binding and degradation is a feature of high-risk HPV E6 not shared by low-risk HPV E6. It also appears that not all of the high-risk HPV E6 interact with the same subset of cellular PDZ proteins. It is unclear how these diverse HPV E6-PDZ protein interactions might account for the shared oncogenic features of high-risk HPV E6, and this point is not discussed in the manuscript. Weaknesses of the manuscript are that the current literature is not cited and that other features of high-risk HPV E6 that might also account for their oncogenic activities are not discussed. Although comparing nucleotide sequence differences is informative, for HPV E6 this comparison does not completely reflect the biology of the proteins. There are high-risk HPV E6-specific protein binding partners other than PDZ domain proteins. Several of these are listed in a useful 2012 review It is established in the HPV literature that small differences in HPV oncoprotein amino acid sequence result in significant differences in interactions with host cellular proteins. This is beautifully illustrated by the structural studies of Gilles Trave and colleagues, who have determined that subtle differences in E6 enable a range of specific interactions with cellular LxxLL-containing proteins. Other recent studies highlight high-risk HPV-specific activities of the E7 oncoprotein. In light of findings like these, this manuscript seems to overstate the claim that the PDZ binding motif is the ‘most notable molecular feature’ distinguishing high- from low-risk HPV. The potential importance of the insert sequence to E6/E7 translation regulation is high and should be tested; the discussion of the importance of the PDZ binding motif should be tempered and put in context with other recent findings.4. HPV16 E6 sequences exhibited variation across the length of the ORF that was similar in high-grade versus control lesions. A possible interpretation of this finding is that the protein sequence in the C-terminus of E6 is not important for oncogenic transformation. How does this fit into the authors’ model? It would be useful to include a discussion of these data.Mirabello and colleagues recently reported an analysis of sequence variants from >5000 HPV16-positive cervical samplesThe phylogenetic trees in Figure 1B might be easier to interpret if they were presented in a linear format. It is difficult to determine where the boundaries between the high-risk/low-risk groupings overlap with the major branch points of the tree.HPV diversity is much greater that that reflected solely in genus alpha. It would be interesting to know whether HPV from other genera have also acquired sequences in this region that might provide some or all of the activities suggested by the authors. Additional points:I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Many references to the current literature were added, and several other features of HR-HPV E6 are now discussed.The suggested references to HR-HPV E6 specific binding partners have been included.The discussion on the importance of the PDZ binding motif in distinguishing HR from LR HPV has been tempered, and the context for its potential relevance has been clarified.We now discuss the recent analysis of 5570 cervical HPV16 genomes (Mirabello and colleagues) and have substantially expanded the discussion of the effect of the insert on E7 production.Figure 1B has been updated, phylogenetic trees are now presented in a linear format. In addition, although the referee have suggested a statistical review of the permutation test that was used in this work to assess the significance of the results, we are confident that this is unnecessary because the statistical technique we used is simple and standard.               We appreciate this highly constructive and helpful review in response to which the following modifications have been made too the article: Auslander and colleagues present a comparative sequence analysis of alpha-papillomavirus genomes. They identify and analyze a short 30-60 nucleotide sequence insert between the E6 and E7 open-reading frames that encodes the PDZ domain in the high-oncogenic risk types and propose that this region also contains sequences that enable coupled termination and reinitiation to facilitate translation of the E6 and E7 proteins. This is an intriguing observation as HPVs are associated with ~5% human cancers and understanding how the viral oncoproteins are expressed is crucial. The proposed hypothesis should be testable and indicates that experiments in which the E6 PDZ domain is mutated in the background of the viral genome should be interpreted carefully.PaVE. There are also many recent publications that describe analyses of PV evolution, oncogenicity and E6 PDZ domains that should be cited. A weakness of the manuscript is that current literature and data sources are not used/and or cited and the data-set used seems incomplete. For example, there are currently 198 officially numbered HPV types, and 442 HPV types in total. A curated data-set of genome sequences for all papillomavirus types sequenced to date is available at There are sequences available for 64 alpha HPV types that have been officially named by the HPV Reference Centre, yet only the genomic sequences of 44 types are listed in Figure S1. The data-set contains many isolates for some HPV types (e.g. 299 isolates of HPV16) yet almost no representatives of the species alpha 2, 4 and 8. The E6 protein of HPV40  has been proposed to contain an ancestral alpha PDZ domain and so the genomes of the alpha-8 species should be closely examined/discussed. Nevertheless, a nucleotide alignment of all 64 alpha-PV nucleotide sequences from PaVE does support the authors’ conclusions that only HR species-5, 6 7, 9 and 11 contain an insert separating the E6 and E7 ORFs.Minor issues:In Figure 1A, alpha-11 is listed in the key for both LR and HR.In Figure 2, the resolution of the sequence images should be improved. The legend for 2B should make it clear that these are just the C-terminal sequences of the E6 polypeptides. In 2C, the symbols and colored blocks should be described.In Figure 3A, the color/bold identification of the motifs and TAA/AUG are not easily visualized in the pdf. Perhaps highlight TAA/AUG by underlines or boxes. Labelling the groups of different HPV species along the left would also be helpful. In 3B, the labelling of the 18S rRNA hairpin (orange text) is confusing. What is 26es7?In Figure S4: clarify in the legend that this is the E6-E7 RNA sequence for each HPV type shown.The reference for IRES (54), I think should be 53.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We appreciate the constructive and very helpful review in response to which the following changes have been made to the manuscript:We now mention the most recent number of  HPV types that have been formally recognized (198), and the reference supporting this has been updated.We added numerous references to recent publications that describe analyses of HPV evolution, oncogenicity, oncoprotein interactions and E6 interactions with PDZ domains.We now include all 64 alpha HPV types from PaVE, and all analyses and figures 1-3 include those HPV strains. Resolution of the new figures has been improved. The legend to Figure 2B has been modified as suggested.The legend to Figure 2C describes all symbols and colored blocks.Figure 3A has been updated to show the motifs clearly and arrange the strains by the order in the phylogenetic tree.Figure 3B has been updated, confusing labelling of 18S rRNA removed.  Although the referee has suggested a statistical review of the permutation test that was used in this work to assess the significance of the results, we are confident that this is unnecessary because the statistical technique we used is simple and standard.