OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.

METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.

RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.

CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.
DNA ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Electrophoresis ; Genetic Vectors ; genetics ; Mutation ; Papillomaviridae ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism

OBJECTIVETo compare the specificity and sensitivity of two genotyping approaches for human papillomavirus (HPV).

METHODHPV DNA was amplified and detected in clinical specimens by polymerase chain reaction in a pair of universal primers MY09/11, and then genotyped with either sequencing method or liquid chip hybridization method (luminex method).

RESULTSequencing method obtained precise genotyping results in single-type HPV infection, while luminex method obtained accurate genotyping results in multiple-type HPV infection.

CONCLUSIONA combined method using both sequencing and luminex method is suitable for the genotyping of HPV-infected specimens.

Base Sequence ; DNA, Viral ; genetics ; Female ; Female Urogenital Diseases ; virology ; Genotype ; Humans ; Oligonucleotide Array Sequence Analysis ; Papillomaviridae ; genetics ; isolation & purification ; Papillomavirus Infections ; virology ; Polymerase Chain Reaction

Epidermodysplasia verruciformis (EV), a rare inherited disease, is believed to be associated with human papillomavirus (HPV) infection. EVER1/2 genes, dendritic cells, T lymphocytes, and the biological characteristics of HPV itself may play roles in the pathogenesis of HPV infection.
Dendritic Cells ; immunology ; Epidermodysplasia Verruciformis ; genetics ; immunology ; virology ; Humans ; Membrane Proteins ; genetics ; Mutation ; Papillomaviridae ; isolation & purification ; pathogenicity ; Papillomavirus Infections ; complications ; T-Lymphocytes ; immunology

OBJECTIVETo develop a new method for the detection of male human papillomavirus (HPV) genotypes and to investigate its clinical application value.

METHODSWith computer assistance and based on the classical common primers MY09/11, modified PGMY09/11 with 23 HPV subtypes for PCR and Genbank data on HPV, we designed probes for the simultaneous detection of 18 high-risk subtypes (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 83 and MM4) and 5 low-risk subtypes (HPV-6, 11, 42, 43 and 44) and fixed them to the special membrane to make a DNA chip. A total of 112 male urethral samples were collected with swabs and studied for the clinical value. Meanwhile the single subtypes of HPV positive were sequenced and the standard samples detected for their sensitivity.

RESULTSOf the total number, 25 samples were found to be HPV positive, 13 single HPV infection and 12 multiple infection. Nine HPV gene subtypes were noted in the samples: 6, 11, 16, 18, 33, 35, 43, 56 and 73, with sensitivity up to 10 copies of HPV DNA.

CONCLUSIONHuman papillomavirus genotyping by the membrane DNA chip is applicable to the diagnosis of male HPV infection as well as to the related epidemic and etiological investigation.

Adult ; Aged ; Base Sequence ; DNA Probes, HPV ; genetics ; DNA, Viral ; genetics ; isolation & purification ; Genotype ; Humans ; In Situ Hybridization ; Male ; Middle Aged ; Molecular Sequence Data ; Papillomaviridae ; genetics ; isolation & purification ; Papillomavirus Infections ; diagnosis ; virology

A considerable number of adult Korean women avoid a Pap smear due to fear and discomfort of the pelvic examination. A reliable but noninvasive and comfortable screening method would considerably increase the participation rate. To evaluate the clinical efficacy of urine-based human papillomavirus (HPV) detection by oligonucleotide microarray, the results of HPV test from matched cervical swab specimens were compared. HPV DNA was detected in 70 of 100 cervical samples. HPV 16 was the most prevalent type (38/70), followed by types 18, 58, 52, 33, 35, 31, and 51. HPV DNA was identified in 47 of 90 urine samples. HPV 16 was the most prevalent type (30/45), followed by types 18, 52, 35, 51, 58, 33, and 56. The HPV detection rates of the cervical swabs increased in accordance with the severity of the cytologic and histologic diagnosis. The type specific agreement of HPV DNA tests between cervical swabs and urine was good in HPV 16 (kappa index=0.64 [95% CI: 0.50-0.79]), 18, 52, and 58 and fair in HPV 33 and 35. We propose that a urine HPV test is a valuable adjunctive method for a conventional Pap smear and can be used in population screening for cervical cancer in countries where it is difficult to obtain colposcopic specimens for cultural or religious reasons.
Vaginal Smears ; Uterine Cervical Neoplasms/*diagnosis ; Papillomaviridae/genetics/*isolation & purification ; Oligonucleotide Array Sequence Analysis ; Middle Aged ; Humans ; Female ; DNA, Viral/*urine ; Cervical Intraepithelial Neoplasia/diagnosis ; Aged ; Adult

In our comparative study of L1 consensus primers with E6 type-specific primers for detection of human papillomavirus (HPVs) by polymerase chain reaction (PCR) in 35 cases of cervical neoplasia, the detection rate by E6 primers (54%; 19/35) was significantly higher than that by L1 primers (25%; 9/35) (p < 0.01). And all specimens HPV-positive with L1 primers were also positive by E6 primers. HPV DNA could be amplified in 36% (9 of 25) of tissue by L1 consensus primers from which beta-globin gene was amplified as compared with 64% (16 of 25) of tissue by E6 type-specific primers. With the L1 consensus primers, 8 cases were positive for HPV-16 and 1 case was positive for HPV-33. These results show that the L1 consensus primers have inferior sensitivity to the E6 type-specific primers for the detection of HPV by PCR. But the L1 consensus primers have great value in making simultaneous detection of various HPV types in a single tube reaction, thus they permit reduction of time and the economic burden of the experiment.
Base Sequence ; DNA, Viral/analysis ; Female ; Humans ; Molecular Sequence Data ; Oligodeoxyribonucleotides ; Papillomaviridae/genetics/*isolation & purification ; *Polymerase Chain Reaction ; Uterine Cervical Neoplasms/*microbiology

Recently, detection of human papillomavirus (HPV)mRNA expression was made possible by in situ hybridization. We described a patient with cervical intraepithelial neoplasia (CIN) 3, showing a distinctive and rare form of co-infection with HPV type 16 and 18. HPV-16 was detected in high grade squamous intraepithelial neoplastic lesion (CIN 3) and HPV-18 was in low grade lesion just adjacent to the HPV-16 infected area. This case suggests that HPV infection may be one of the most responsible causative agents producing malignant transformation and two distinctive HPV types can also simultaneously infect the squamous epithelium of the uterine cervix.
Adult ; Cervical Intraepithelial Neoplasia/*microbiology ; Cervix Uteri/microbiology ; Female ; Humans ; In Situ Hybridization ; Papillomaviridae/genetics/*isolation & purification ; Papillomavirus Infections/*microbiology ; Tumor Virus Infections/complications/*microbiology ; Uterine Cervical Neoplasms/*microbiology

OBJECTIVETo study the circulation, distribution, and genomic diversity of HPVs in common warts in Beijing area of China.

METHODSForty eight patients with pathologically diagnosed common warts were screened for the presence of HPV with HPV type-specific PCR and direct sequencing analysis. The genomic diversity of HPVs prevalent in Chinese patients was analyzed based on LCR.

RESULTSForty one (85.5%) samples were positive for HPV DNA, 13 (31.7%)--HPV-57, 12 (29.3%)--HPV-1a, 7 (17%)--HPV-27 and 5(12.2%)--HPV-2a. Four cases were infected with two different HPV types, two (4.9%) with HPV-1a and HPV-27, one (2.4%) with HPV-1 and HPV-57 and one (2.4%) with HPV-27 and HPV-57. In contrast to the prevalence of single strain of novel HPV-57 variant and HPV-1 prototype, two HPV-2 and three HPV-27 novel variants were found to circulate in Beijing.

CONCLUSIONHPV-1, -2, -27 and -57 are predominantly prevalent in patients with common warts in Beijing.

Adolescent ; Adult ; Aged ; China ; epidemiology ; DNA, Viral ; Female ; Genetic Variation ; Humans ; Male ; Middle Aged ; Papillomaviridae ; classification ; genetics ; isolation & purification ; Phylogeny ; Prevalence ; Warts ; epidemiology ; virology

OBJECTIVEThis study was designed to determine the prevalence of oncogenic human papillomavirus (HPV) in cervical infections in Beijing, China, and to investigate the odds ratio (OR) of HPV single and multiple infections in abnormal cytology.

METHODSA total of 19,018 specimens from outpatients in the department of obstetric and gynecology were collected. They were detected using high-risk HPV genotyping real-time polymerase chain reaction (PCR) kit and analyzed by ThinPrep cytology test for cervical pathological diagnosis. HPV prevalence, age-specific prevalence, and OR of each type of HPV in abnormal cytology were analyzed.

RESULTSOverall, 19.1% (3,623/19,018) of the individuals were positive for HPV infection, 14.9% (2,833/19,018) were positive for a single HPV type, and 4.2% (790/19,018) were positive for multiple types. Among the 3,623 HPV-positive individuals, the most predominant HPV types were HPV52 (4.4%, 834/19,018), HPV16 (3.7%, 710/19,018), and HPV58 (3.4%, 644/19,018). The OR of multiple infections and single infection differed significantly among disease severities. The OR of dual infection was higher than that of each of the two single infection types, respectively.

CONCLUSIONHPV prevalence in the outpatients was 19.1%, and the most predominant HPV types in the study were HPV52, HPV16, and HPV58. Women with multiple infectionswere more likely to have abnormal cytology.

Adolescent ; Adult ; Aged ; Beijing ; Female ; Genotype ; Humans ; Middle Aged ; Papillomaviridae ; classification ; genetics ; isolation & purification ; Papillomavirus Infections ; pathology ; virology ; Uterine Cervical Neoplasms ; pathology ; virology ; Young Adult