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 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 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 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

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

OBJECTIVEHuman papillomavirus (HPV) commonly exists in healthy individuals, but its prevalence in the pubic hair follicles is not yet clear, nor is the relationship between HPV infection in the pubic hair follicles and the recurrence of genital warts in men. This study aimed to investigate HPV infection in the pubic hair follicles of healthy men and patients with genital warts, and to look into the correlation of HPV infection with recurrent genital warts.

METHODSWe included in this study 122 healthy men aged 21-80 years and 86 male patients with genital warts aged 24-61 years, detected HPV in their pubic hair follicles by PCR, and made comparative analysis of the data obtained from the two groups.

RESULTSThe positive rate of HPV in the pubic hair follicles of the healthy males was 17.21% (21/122), including 15 cases of HPV6, 4 HPV11, 1 non-HPV6/11 and 1 the mixed type (both HPV6 and HPV11), while that of the genital wart patients was 32.55% (28/86), including 17 cases of HPV6, 7 HPV11, 2 non-HPV6/11 and 2 the mixed type.

CONCLUSIONThe incidence of HPV infection is higher in patients with genital warts than in healthy men, while the types of HPV involved are basically the same in the two groups, mainly HPV6 and HPV11.

Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Condylomata Acuminata ; epidemiology ; virology ; Hair Follicle ; virology ; Humans ; Incidence ; Male ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; epidemiology ; Young Adult

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