We wished to screen the cell line that stably expresses the HPV18E5 protein, and to ascertain the influence of HPV18E5 protein on cell proliferation and the cell cycle. The HPV18E5 gene was amplified by the polymerase chain reaction. Then, the His-tag pSecTag-HPV18E5 eukaryotic expression vector was constructed by digestion ligation and connection. The recombinant plasmid was transfected into Balb/c3T3 cells with lipofectamine, and positive cell lines were screened by a culture medium containing bleomycin. HPV18E5 expression in cells was confirmed by western blotting and immuno-enzymatic methods. The influence of HPV18E5 on cell proliferation and the cell cycle were detected by Cell Counting Kit-8 and flow cytometry, respectively. The pSecTag-HPV18E5 eukaryotic expression vector was constructed. After 21-day selection in a culture medium containing 400 μg/mL bleomycin, stably expressing HPV18E5 protein cells were harvested. Compared with control groups, cell proliferation in HPV18E5 stably expressed cells was obviously increased, as was the S phase in the cell cycle. Our results suggested that HPV18E5 influences cell proliferation and the cell cycle. Our study has laid the foundation of the biologic properties of HPV18E5 protein, which will aid further studies on the mechanism of action of carcinogenesis.
Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Human papillomavirus 18 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; physiopathology ; virology ; Transfection

BACKGROUND: We evaluated the performance of various commercial assays for the molecular detection of human papillomavirus (HPV); the recently developed AdvanSure HPV Screening real-time PCR assay (AdvanSure PCR) and the Abbott RealTime High Risk HPV PCR assay (Abbott PCR) were compared with the Hybrid Capture 2 HPV DNA Test (HC2). METHODS: All 3 tests were performed on 177 samples, and any sample that showed a discrepancy in any of the 3 tests was genotyped using INNO-LiPA HPV genotyping and/or sequencing. On the basis of these results, we obtained a consensus HPV result, and the performance of each test was evaluated. We also evaluated high-risk HPV 16/18 detection by using the 2 real-time PCR assays. RESULTS: Among the 177 samples, 65 were negative and 75 were positive in all 3 assays; however, the results of the 3 assays with 37 samples were discrepant. Compared with the consensus HPV result, the sensitivities and specificities of HC2, AdvanSure PCR, and Abbott PCR were 97.6%, 91.7%, and 86.9% and 83.9%, 98.8%, and 100.0%, respectively. For HPV type 16/18 detection, the concordance rate between the AdvanSure PCR and Abbott PCR assays was 98.3%; however, 3 samples were discrepant (positive in AdvanSure PCR and negative in Abbott PCR) and were confirmed as HPV type 16 by INNO-LiPA genotyping and/or sequencing. CONCLUSIONS: For HPV detection, the AdvanSure HPV Screening real-time PCR assay and the Abbott PCR assay are less sensitive but more specific than the HC2 assay, but can simultaneously differentiate type 16/18 HPV from other types.
Adult ; Aged ; Cervix Uteri/pathology/virology ; DNA, Viral/analysis ; Female ; Genotype ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Middle Aged ; Papillomaviridae/*genetics/isolation & purification ; Papillomavirus Infections/*diagnosis/pathology/virology ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Young Adult

OBJECTIVE: Infection with high-risk genotypes of human papillomavirus (HR-HPV) is the major cause of invasive cervical cancers. HPV-16 and HPV-18 are known to be responsible for two-thirds of all invasive cervical carcinomas, followed by HPV-45, -31, and -33. Current guidelines only differentiate HPV-16/18 (+) by recommending direct colposcopy for treatment. We tried to evaluate whether there are differences in risk among 12 non-16/18 HR-HPV genotypes in this study. METHODS: The pathology archive database records of 1,102 consecutive gynecologic patients, who had results for cervical cytology and histology and for HPV testing, as determined by HPV 9G DNA chip, were reviewed. RESULTS: Among the 1,102 patients, 346 were non-16/18 HR-HPV (+) and 231 were HPV-16/18 (+). We calculated the odds ratios for ≥cervical intraepithelial neoplasia 2 (CIN 2) of 14 groups of each HR-HPV genotype compared with a group of HR-HPV (–) patients. Based on the odds ratio of each genotype, we divided patients with non-16/18 HR-HPV genotypes (+) into two groups: HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+). The age-adjusted odds ratios for ≥CIN 2 of the HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+) groups compared with a HR-HPV (–) group were 11.9 (95% CI, 7.6 to 18.8; p<0.001) and 2.4 (95% CI, 1.4 to 4.3; p<0.001), respectively, while that of the HPV-16/18 (+) group was 18.1 (95% CI, 11.6 to 28.3; p=0.003). CONCLUSION: The 12 non-16/18 HR-HPV genotypes can be further categorized (HPV-31/33/35/45/52/58 vs. HPV-39/51/56/59/66/68) by risk stratification. The HPV-31/33/35/45/52/58 genotypes might need more aggressive action. Large scale clinical trials or cohort studies are necessary to confirm our suggestion.
Adult ; Cervical Intraepithelial Neoplasia/*virology ; Colposcopy ; DNA, Viral/analysis ; Female ; *Genotype ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Middle Aged ; Papanicolaou Test ; Papillomaviridae/*genetics ; Papillomavirus Infections/*virology ; Risk Factors ; Uterine Cervical Neoplasms/virology ; Vaginal Smears

Objective: To examine the prevalence and frequencies of human papillomavirus (HPV) genotypes in cervical adenocarcinoma in situ (AIS). Methods: The cases of cervical AIS with concurrent tests of cytology and HPV typing from January 2007 to February 2020 in the Obstetrics and Gynecology Hospital of Fudan University were collected and analyzed. Results: A total of 478 cases of cervical AIS were obtained. The average age of the patients was 39.4 years (range, 19-81 years). The largest age group was 30-39 years (44.8%), followed by 40-49 years (34.7%). Among the 478 patients, 355 underwent high-risk HPV (hrHPV) testing and had a hrHPV-positive rate of 93.8%. Of the 355 patients, 277 also underwent HPV typing and were mostly positive for either or both HPV16 and HPV18 (93.1%), with 55.6% positive for HPV18 and 48.7% positive for HPV16. Among the 478 cases, 266 cases (55.6%) were diagnosed with both AIS and squamous intraepithelial lesion (SIL), while 212 cases (44.4%) were diagnosed with only AIS. Patients infected with HPV16 in the AIS and SIL group significantly outnumbered those in the AIS alone group (P<0.05). Moreover, the rate of positive cytology was 55.9% (167/299 cases), while that of negative cytology was 44.1% (132/299). Among the 109 patients with negative cytology results and co-tested hrHPV, there were 101 HPV-positive cases (92.7%), of which 88 cases were subject to HPV typing and showed an HPV16/18 positive rate of 94.3% (83/88 cases). Conclusions: The combination of HPV typing and cytological screening can maximize the detection rate of cervical AIS, and should continue to be utilized, ideally on a larger scale, in the future.
Adenocarcinoma in Situ/epidemiology* ; Adult ; Aged ; Aged, 80 and over ; Female ; Human papillomavirus 16/genetics* ; Human papillomavirus 18/genetics* ; Humans ; Middle Aged ; Papillomaviridae/genetics* ; Papillomavirus Infections/diagnosis* ; Prevalence ; Uterine Cervical Neoplasms/pathology* ; Young Adult

OBJECTIVETo evaluate the diagnostic applicability of human papillomavirus (HPV) liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong province.

METHODSTo detect HPV genotypes on a 96-well plate with the liquid chip which can simultaneously detect and identify 26 common HPV genotypes in a total of 2925 cervical scrapes obtained from gynecological outpatients as well as to analyze the relationship between HPV types and different cervical diseases by studying the distribution of HPV genotypes and pathologic diagnosis.

RESULTSAmong 639 cases who performed pathologic/cytological and histological diagnoses, 184 cases are in group of normal cytology, 266 cases in group of, 77 cases in group of cervical intra-epithelial neoplasia (CIN) I, 7 cases in group of CIN I - II, 46 cases in group of CIN I - II, 46 cases in group of CIN I - II and 13 cases in group of cervical cancer. The overall incidence of HPV in our samples is 36.0% (1054/2925) and 23 types of all 26 types on liquid chip are found. The most common genotypes found are HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18 (8.87%) and HPV-11 (6.94%). Among all the positive types, 87.32% are high-risk HPV and 13.68% are low-risk HPV genotypes. Both single and multiple types are easily identified, showing 66.22% ( n = 698) single type and 33.78% ( n = 356) multiple types. Of all the 1054 HPV-positive cases, 261 (24.8%) is occupied by women 21 to 25 years of age and progressively lower by older age groups, reaching 4.9% by women between 51 to 67 years old. The incidence of HPV in our samples is 23.37%, 33.08%, 54.54%, 57.14%, 82.61%, 91.30% and 100% for normal cytology, inflammation,CIN I ,CIN I - II, CIN II ,CIN III, and carcinomas specimens, respectively. Infections with more that one virus are common, accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I - II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II, III are 88.57%, 76.63%, 68.89% and 92.16% respectively. Conclusion The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. The HPV-positive rate increased along with the increase of grading on cervical lesions. There are more younger women among all the HPV-positive ones. Multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.

Adolescent ; Adult ; Aged ; Cervical Intraepithelial Neoplasia ; epidemiology ; virology ; China ; epidemiology ; Female ; Gammapapillomavirus ; classification ; genetics ; Genotype ; Human papillomavirus 11 ; classification ; genetics ; Human papillomavirus 16 ; classification ; genetics ; Human papillomavirus 18 ; classification ; genetics ; Human papillomavirus 6 ; classification ; genetics ; Humans ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Papillomaviridae ; classification ; genetics ; Papillomavirus Infections ; epidemiology ; virology ; Uterine Cervical Neoplasms ; epidemiology ; virology ; Young Adult

Female ; Human papillomavirus 18 ; physiology ; Humans ; Papillomavirus Infections ; complications ; Risk Factors ; Uterine Cervical Neoplasms ; epidemiology ; etiology ; pathology ; virology ; Virus Integration ; genetics ; physiology

OBJECTIVESTo evaluate the significance of p16(INK4A) protein expression and positivity for HPV DNA in distinguishing between endocervical and endometrial adenocarcinoma.

METHODSExpression of p16(INK4A) protein in 30 cases of endocervical adenocarcinoma and 10 cases of endometrial adenocarcinoma was assessed by immunohistochemistry. In-situ hybridization for human papillomavirus (HPV) DNA was also performed in 20 cases of endocervical adenocarcinoma and 10 cases of endometrial adenocarcinoma.

RESULTSThe positive rate for p16(INK4A) in endocervical adenocarcinoma was 70% (21/30), as compared with 30% (3/10) in endometrial adenocarcinoma. The tumor cells in endocervical adenocarcinoma showed diffuse and strong expression of p16(INK4A) protein with both cytoplasmic and nuclear staining. In contrast, the endometrial adenocarcinoma cells showed patchy and weak expression of p16(INK4A). On the other hand, HPV DNA (type 16 or 18) was detected by in-situ hybridization in 9 (45%) of the 20 cases of endocervical adenocarcinoma and none of the 10 cases of endometrial adenocarcinoma.

CONCLUSIONSThe expression of p16(INK4A) protein is significantly higher in endocervical adenocarcinoma than in endometrial adenocarcinoma. This expression pattern can serve as a useful immunohistochemical marker in the differential diagnosis. p16(INK4A) protein immunohistochemistry appears to be more sensitive than HPV DNA testing in distinguishing between endocervical and endometrial adenocarcinoma, especially in biopsy or curettage specimens.

Adenocarcinoma ; diagnosis ; genetics ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA, Viral ; analysis ; Endometrial Neoplasms ; diagnosis ; genetics ; virology ; Female ; Gene Expression Regulation, Neoplastic ; Human papillomavirus 16 ; genetics ; Human papillomavirus 18 ; genetics ; Humans ; In Situ Hybridization ; Uterine Cervical Neoplasms ; diagnosis ; genetics ; virology

OBJECTIVEThe Human papillomavirus type 18L1 (HPV18L1) gene was synthesized by overlapping PCR after optimization using plant preferred codons.

METHODSThe gene sequences of HPV18L1 were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. The target sequence was selected and modified using plant preferred codons by the Synthetic Gene Designer and JCat (Java Codon Adaptation Tool) with the addition of a His-tag to the C-terminus to construct the full-length modified HPV18L1 (mHPV18L1). mHPV18L1 was divided into 5 large segments, namely LS1 to LS5, with sizes ranging from 204 to 477 bp. Forty-three small oligonucleotide fragments with sizes of 57-59 bp and 6 pairs of primers were designed and synthesized. mHPV18L1 was amplified by overlapping PCR and subcloned into pMD18-T vector. The recombinant plasmid was identified by restriction enzymes digestion and sequencing.

RESULTSmHPV18L1 was successfully assembled using overlapping PCR. The results of digestion with restriction enzymes and PCR amplification confirmed that the recombinant vector pMD18T- mHPV18L1 contained the inserts with expected size of 1749 bp. mHPV18L1 sequence was confirmed by sequencing.

CONCLUSIONmHPV18L1 with plant preferred codons and the recombinant vector pMD18T- mHPV18L1 have been obtained.

Base Sequence ; Capsid Proteins ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genes, Plant ; genetics ; Genes, Synthetic ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 18 ; genetics ; Molecular Sequence Data ; Papillomavirus Vaccines ; genetics ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; genetics

OBJECTIVETo investigate human papillomavirus (HPV) infection and genotyping in patients with cervical cancer in Guangzhou in the last 3 decades.

METHODSHPV L1 gene fragment in paraffin-embedded cervical cancer samples was amplified by HPV-specific PCR with consensus primers, and typing of HPV strains was performed on the basis of sequence analysis of the PCR products.

RESULTSThe positivity rates of HPV DNA was 26.2% in the 99 cases of cervical cancer. Five HPV genotypes were identified including HPV16, 18, 33, 52 and 58.

CONCLUSIONHPV16, 58 and 33 are the most common genotypes of HPV, accounting for over 88.4% in the total infected cases, suggesting that the HPV genotypes closely related to cervical cancer is more centralized in Guangzhou.

China ; epidemiology ; DNA, Viral ; analysis ; Female ; Genotype ; Human papillomavirus 16 ; isolation & purification ; Human papillomavirus 18 ; isolation & purification ; Humans ; Papillomaviridae ; genetics ; Papillomavirus Infections ; epidemiology ; virology ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; epidemiology ; pathology ; virology

Objective: To evaluate the performance of point-of-care testing for cervical cancer and precancerous lesions screening. Methods: In September 2020, 197 and 273 women were selected by using simple random sampling method from "self-sampling" cohort and "physician-sampling" cohort established in Xiangyuan county, Shanxi Province, China, respectively. Cervical exfoliated cells were collected by women themselves or gynecologists. All samples were detected by POCT and women with positive result were directly referred for colposcopy. Subsequently, all the samples were detected by careHPV and PCR test. Colposcopy and punch biopsy were performed for women with POCT negative but careHPV or PCR test positive at another visit. Using histopathological diagnosis as the gold standard, we calculated sensitivity, specificity and drew the receiver operating characteristic (ROC) curves. The accuracy of POCT was analyzed and compared to that of careHPV and conventional PCR test in cervical cancer and precancerous lesions screening. Results: The median (Q₁ , Q₃) age of 470 women was 51 (45, 57) years old. Based on self-sampling, the sensitivity and specificity of POCT for CIN2+ were 100.00% (95%CI: 56.56%-100.00%) and 28.95% (95%CI: 22.97%-35.76%), respectively. Compared with POCT, POCT HPV16/18 test had similar sensitivity and higher specificity of 89.47% (95%CI: 84.30%-93.08%). Self-sampling POCT HPV16/18 test had an AUC of 0.947 (95%CI:0.910-0.985), which was higher than that of careHPV and PCR test. Physician-sampling POCT test had 100.00% sensitivity (95%CI: 64.57%-100.00%) and 55.85% specificity (95%CI: 49.83%-61.70%) for detecting CIN2+. POCT HPV16/18 test had lower sensitivity (71.43%, 95%CI: 35.90%-91.76%) and higher specificity (92.45%, 95%CI: 88.63%-95.06%). POCT HPV16/18 test generally showed similar AUC on both self-collected samples and clinician-collected samples (0.947 vs 0.819, P=0.217). Conclusion: POCT HPV16/18 test is an effective method with relatively high sensitivity and specificity for cervical cancer screening.
Cervical Intraepithelial Neoplasia/diagnosis* ; Colposcopy ; Early Detection of Cancer/methods* ; Female ; Human papillomavirus 16/genetics* ; Human papillomavirus 18 ; Humans ; Mass Screening/methods* ; Papillomaviridae ; Papillomavirus Infections/diagnosis* ; Point-of-Care Testing ; Pregnancy ; Sensitivity and Specificity ; Uterine Cervical Neoplasms