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

OBJECTIVETo detect and type human papilloma virus (HPV) in the warts of oral mucosa from HIV-positive patients, and better understand the biological characters of these oral warts.

METHODSPolymerase chain reaction (PCR) was used to detect and type HPV infection by consensus HPV primers Gp5+/Gp6+ and specific HPV primers (HPV6/11, 16, 18, 31, 33) in 34 cases of oral mucosa warts from HIV-positive patients.

RESULTSThe HPV infection rate was 88.2% by consensus HPV primers Gp5+/Gp6+; the HPV infection rate of HPV6/11, 16, 18, 31 was respectively 47.06%, 11.67%; 2.94%, and 5.88% by specific HPV primers.

CONCLUSIONMost lesions of oral warts from HIV-positive patients are associated with the infection of HPV. The low risk HPV6/11 infection is more common than the high risk HPV16, 18, 31.

HIV Infections ; virology ; HIV Seropositivity ; Humans ; Mouth Diseases ; virology ; Mouth Mucosa ; pathology ; virology ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; diagnosis

OBJECTIVETo identify and assess multiple human papillomavirus types in condyloma acuminatum lesions from patients with genital warts in Beijing area, and compare different features between otherwise healthy and immunosuppressed patients.

METHODSPCR, RFLP and nucleotide sequencing analysis were used to determine HPV types from individual lesions.

RESULTSThe predominant type from other healthy patients was HPV6, secondly HPV11. The mean age of patients infected by HPV6 was lower than that of HPV11 and HPV6 + 11. While lesions from immunosuppressed patients were often contained HPV11 or mixed with HPV6. Besides, HPV types 16 and 53 were detected from infected lesions than other HPV types.

CONCLUSIONSHPV6 was the major pathogen of condyloma acuminatum, but infected patients were at lower ages. While HPV11 was most often detected from immunosuppressed patients. As a low risk virus in normal genital tract, HPV53 also could be a pathogen in genital warts.

Adult ; Condylomata Acuminata ; virology ; Female ; Humans ; Male ; Papillomaviridae ; classification ; isolation & purification ; Papillomavirus Infections ; Tumor Virus Infections ; Warts ; virology

OBJECTIVETo further understand the clinicopathological, ultrastructural and molecular features of penile pseudoangiosarcomatous squamous cell carcinoma (PASCC), and improve its diagnosis and treatment.

METHODSA 47-year-old male patient with penile PASCC was reported and the relevant literature reviewed. The main clinical manifestations of the patient were a typical surface ulceration with hemorrhage and purulent secretion with a foul smell, a papillary mass about 5.0 cm x 5.0 cm x 4.0 cm for 1 year on the foreskin of the penis, and 3 enlarged bilateral inguinal lymph nodes. CT scanning showed no enlarged lymph nodes in the abdomen and pelvis, and X-ray examination revealed no abnormality in the chest.

RESULTSThe diagnosis was established by biopsy. Partial penectomy and bilateral inguinal lymphadenectomy (T2N2M0) were performed, followed by adjuvant pelvic radiotherapy. Two months later, total penectomy was necessitated by penile flap necrosis and local recurrence. Eleven months after the first surgery, the patient died of extensive metastasis to the pelvis and lungs. Under the light microsope, the tumor was mainly composed of vessel-like lacunar reticularis spindle cells and a few local squamous cancer cells. Careful examination revealed some focal areas with evident transition from squamous nests to the more acantholytic areas extending towards the pseudoangiosarcomatous spaces. Pathogenetically, it appeared to be the variant of acantholytic squamous cell carcinoma. Immunohistochemically, most tumor cells were strongly positive for keratin (AE1/AE3) and focally positive for EMA, with the typical squamous cells focally positive for 34betaE12 and vimentin. The vessels that proliferated in the tumor were decorated by CD31, CD34 and factor VIII-related antigens, but the tumor cells were negative for HMB45, SMA, Desmin and CEA. HPV DNA (HPVpan, HPV6B/11, HPV16/18, HPV31/33) was not detected by in situ hybridization in the primary and metastatic tumors.

CONCLUSIONPASCC is a specific and extremely rare subtype of penile SCC with dramatic similarity to angiosarcoma under the microscope, with poor prognosis. Its diagnosis depends on histopathological, immunohistochemical and ultrastructural studies. Such a presentation underscores the importance of timely consultation, early diagnosis and prompt treatment.

Carcinoma, Squamous Cell ; pathology ; virology ; Humans ; Male ; Middle Aged ; Papillomaviridae ; isolation & purification ; Penile Neoplasms ; pathology ; virology ; Penis ; pathology ; virology

OBJECTIVETo investigate the infection rate and subtypes of human papilloma virus(HPV) in patients with oropharyngeal squamous cell carcinoma (OSCC) and analyze the clinicopathologic features of patients with or without HPV infection.

METHODSA total of 66 biopsy or surgical specimens of OSCC archived in the Pathology Department of Chinese Academy of Medical Sciences were analyzed by polymerase chain reaction (PCR), and the generic amplification products were detected by DNA enzyme immunoassay (DEIA) and typed by reverse hybridization line probe assay.

RESULTSHPV-DNA was detected in 11 (16.7%) of all specimens. Among them, 7 were infected with HPV-16,and the remaining 4 patients were infected with HPV-16/11, HPV-35, HPV-58/52, and HPV-33/52/54, respectively. HPV-16 was detected in 72.7% of all positive specimens. There were more females in HPV-positive group than HPV-negative group (36.4% vs. 1.8%,P=0.002). Patients with HPV-positive tumors were more likely to be non-smokers (36.4% vs. 0,p=0.001) and non-drinkers (45.5% vs. 1.8%,p=0.001) than those with HPV-negative tumors. The proportion of moderately or poorly differentiated tumors was higher in HPV-positive patients than HPV-negative patients (81.8% vs. 63.7%), although without statistical significance (p=0.409). No difference was observed in T classification, N classification, and overall tumor stage.

CONCLUSIONSHPV infection rate was 16.7% in this cohort. HPV-positive OSCC has its unique etiologic and clinicopathological characteristics.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; virology ; DNA, Viral ; isolation & purification ; Female ; Humans ; Male ; Middle Aged ; Oropharyngeal Neoplasms ; virology ; Papillomaviridae ; classification ; isolation & purification ; Papillomavirus Infections ; virology

Objective To analyze the infection status of human papilloma virus (HPV),Ureaplasma urealyticum (UU),Chlamydia trachomatis (CT),and Neisseria gonorrhoeae (NG) in clinical patients.Methods The laboratory specimens including urine,urethral swabs,and cervical swabs from 870 patients from January 1st 2014 to December 31st 2017 were retrospectively analyzed. HPV-DNA was detected by multiplex fluorescent PCR,and the UU-RNA,CT-RNA,and NG-RNA were determined by isothermal nucleic acid amplification. The positive rate of each pathogen and the distribution of positive rate between male and female patients were calculated. The samples were further divided into HPV-positive group and HPV-negative group,and the positive rates of UU-RNA,CT-RNA,and NG-RNA in these two groups were compared.Results The highest positive rate was 53.68%(467/870) for UU-RNA,followed by HPV-DNA [32.41%(282/870) ]and NG-RNA [2.18%(19/870)]. The total positive rate of high-risk (HR)-HPV(subtypes:16,18,31,33,35,39,45,51,52,56,58,59,and 68) [31.52%(209/663)]and UU in female patients [60.93%(404/663)] was significantly higher than that in male patients [17.39%(36/207),30.34%(63/207)](both P<0.001). The male patients had significantly higher CT positive rate in HR-HPV-positive group than in HR-HPV-negative group [22.58%(7/31) vs. 4.54%(8/176)](P<0.001). The female patients had significantly higher CT positive rate in HR-HPV-positive group than in HR-HPV-negative group [10.5%(21/200) vs. 5.61%(26/463)](P=0.024). The UU-RNA positive rate of females in the low-risk (LR)-HPV (subtypes:6 and 11) positive group was significantly higher than that in LR-HPV negative group [70.83%(34/48) vs.2.11%(13/615)](P<0.001).Conclusions Women are more susceptible to HR-HPV and UU infections. HR-HPV-positive patients are more likely to experience CT infection. In contrast,co-infection with UU is more common in LR-HPV-positive females.
Chlamydia Infections ; diagnosis ; epidemiology ; Chlamydia trachomatis ; isolation & purification ; Female ; Gonorrhea ; diagnosis ; epidemiology ; Humans ; Male ; Neisseria gonorrhoeae ; isolation & purification ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; diagnosis ; epidemiology ; Retrospective Studies ; Ureaplasma Infections ; diagnosis ; epidemiology ; Ureaplasma urealyticum ; isolation & purification

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

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 rate of concordance between positive cervical cytologic interpretation based on liquid-based preparations and subsequent histologic diagnosis.

METHODSLiquid-based cervical cytology (SurePath) was carried out in 15,393 patients. Cases with the cytologic diagnosis of epithelial cell abnormality were further investigated by human papillomavirus (HPV) DNA testing (Hybrid Capture II) for the high-risk HPV DNA, colposcopic examination and multiple cervical biopsies. The 2001 version of The Bethesda System for reporting gynecologic cytology was applied. Positive cytologic diagnosis included atypical squamous cells of undetermined significance (ASC-US) or above. Histologic correlation was available in some of the patients. The rate of concordance between positive cytologic interpretation and subsequent histologic diagnosis was calculated.

RESULTSAmongst the 15,393 liquid-based cytology cases studied, the cytologic diagnosis of 7 cases squamous cell carcinoma were all concordance with histologic diagnosis, while the rates for high-grade and low-grade squamous intraepithelial lesions were 93.6% (103/110) and 82.0% (443/540), respectively. A positive correlation was also demonstrated between detection of high-risk HPV DNA, positive cytologic interpretation and positive histologic diagnosis.

CONCLUSIONLiquid-based cytologic preparation, when coupled with standardized reporting using the Bethesda System 2001, can achieve a high diagnostic accuracy of cervical epithelial abnormalities.

Carcinoma, Squamous Cell ; pathology ; Cervical Intraepithelial Neoplasia ; pathology ; Cervix Uteri ; pathology ; DNA, Viral ; analysis ; Female ; Humans ; Papillomaviridae ; isolation & purification ; Retrospective Studies ; Uterine Cervical Neoplasms ; pathology ; Vaginal Smears ; methods

BACKGROUNDHuman papillomavirus (HPV) is believed to be the most common sexually transmitted infection. However, little is known about the prevalence and distribution of HPV types in China. We aimed to assess the prevalence and the distribution of HPV types as well as risks for abnormal cervical cytology in women who reside in the Tibetan Autonomous Region of China.

METHODSA cross-sectional study was performed involving a sample of 3036 women. An epidemiological questionnaire was applied and cervical specimens were obtained for liquid-based cytology and HPV DNA detection. Statistical analysis included chi-square and Logistic regression model.

RESULTSIn this population, 3.66% (111/3036) had atypical squamous cells of undetermined significance (ASCUS), 1.45% (44/3036) low-grade squamous intraepithelial lesions (LSIL) and 1.09% (33/3036) had high-grade squamous intraepithelial lesions (HSIL). Tibetan women (5.74%, 137/2387) exhibited lower abnormal cytology rates than non-Tibetan women (8.01%, 52/649, P = 0.03). The overall prevalence of HPV infection was 9.19% (279/3036). We failed to identify any differences in HPV prevalence by age. In the groups with normal, ASCUS, LSIL and HSIL, the overall HPV prevalences were 7.41% (211/2847), 24.32% (27/111), 56.82% (25/44) and 45.45% (15/33), respectively. HPV 16 (1.52%, 46/3036) was the most common type, and was also the most prevalent in women with ASCUS (8.11%, 9/111) and HSIL (15.15%, 5/33). The most common HPV type for Tibetan women was HPV 16 (1.42%, 34/2387), whereas for non-Tibetan individuals it was HPV 33 (2.31%, 15/649). Of the 279 HPV-infected women, 40 individuals (14.34%) presented with multiple HPV positivity. Women who had two pregnancies were more likely to have abnormal cytology smear (OR = 1.67; 95%CI: 1.07 - 2.61).

CONCLUSIONSA low prevalence of HPV positivity was observed in women who reside in the Tibetan Autonomous Region of China. The prevalence of abnormal cervical cytology and HPV type distributions were different between Tibetan and non-Tibetan women.

Adult ; Age Distribution ; Cervix Uteri ; virology ; China ; Cross-Sectional Studies ; Female ; Humans ; Middle Aged ; Papillomaviridae ; classification ; isolation & purification ; Papillomavirus Infections ; epidemiology ; Risk Factors ; Surveys and Questionnaires