1.Mutations in various functional domains of HPV2 E2 protein inhibit the transcriptional depression activities.
Chen GAO ; Yan-Jun LEI ; Hui-Ying JIANG ; Qi SHI ; Chan TIAN ; Jun HAN ; Xiao-Ping DONG
Chinese Journal of Experimental and Clinical Virology 2011;25(3):164-166
OBJECTIVETo study the potential transcriptional depression activities of HPV2 E2 proteins with mutations in different functional domains.
METHODSThe primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptional repression activities of the E2 mutants were evaluated by detection of CAT expression values.
RESULTSCompared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptional repressive activities. The point mutations in the transactivation domain (nt 3037), the internal hinge region (nt 3387) and DNA binding domain (nt 3697) showed remarkable inhibition on its transcriptional depression function.
CONCLUSIONThe transcriptional regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.
HeLa Cells ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; genetics
2.Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris.
Ping-chuan LI ; Xiao-guang ZHANG ; Ling ZHOU ; Yi ZENG
Chinese Journal of Experimental and Clinical Virology 2003;17(4):310-314
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
3.Detection and typing of human papillomavirus by a GeXP based multiplex PCR assay.
Chun-bin LU ; Meng-jie YANG ; Le LUO ; Miao WANG ; Xue-jun MA
Chinese Journal of Experimental and Clinical Virology 2011;25(1):69-72
OBJECTIVETo establish a new and rapid GeXP based multiplex PCR assay for the detection and typing of human papillomavirus 6, 11, 31, 33 and 52.
METHODSNucleotide sequences of HPV6, HPV11, HPV31, HPV33 and HPV52 from NCBI were obtained and compared. Genotype-specific primers were then designed and the sensitivity and specificity of multiple PCR assay was evaluated. Optimized assay was further validated with 30 clinical specimens collected from the cervical secretions of patients.
RESULTSA GeXP based multiplex PCR was developed for sensitive detection and reliable differentiation of five HPV genotypes (HPV6, 11, 31, 33 and 52),
CONCLUSIONA GeXP based multiplex PCR assay is demonstrated to be a new and rapid technique for simultaneous detection and typing of 5 different human papillomaviruses.
Adult ; Female ; Humans ; Middle Aged ; Papillomaviridae ; classification ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods
4.Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells.
Ruslana KADZE ; Philip J CHAN ; John D JACOBSON ; Johannah U CORSELLI ; Alan KING
Asian Journal of Andrology 2002;4(3):169-173
AIMTo pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells.
METHODSCumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined.
RESULTSOver half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups.
CONCLUSIONSperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.
DNA, Viral ; pharmacokinetics ; Humans ; Male ; Papillomaviridae ; genetics ; Spermatozoa ; physiology ; Temperature ; Transfection ; methods
5.Characteristics of cervical microecology in late reproductive-age women with different grades of cervical lesions.
Qingzhi ZHAI ; Tengjie REN ; Yurong FU ; Zhe ZHANG ; Li'an LI ; Yali LI ; Yuanguang MENG
Journal of Southern Medical University 2020;40(12):1768-1775
OBJECTIVE:
To analyze the characteristics of cervical microecology in late reproductive-age women with cervical lesions and explore new methods for preventing cervical lesions.
METHODS:
Cervical smears were obtained from a total of 147 women of late reproductive age, including 24 with high-risk HPV infection (HR-HPV), 27 with low-grade squamous intra-epithelial lesions (LSIL), 36 with high-grade squamous intra-epithelial lesions (HSIL), 35 with cervical cancer (CC) and 25 healthy women. llumina MiSeq sequencing of V3-V4 region of the 16S rRNA gene amplicons was used to characterize the vaginal microbiota of the women. OTUs analysis of the valid data was performed, and the α-diversity (Chao1, Simpson's Index and Shannon Index) and β-diversity (T-test, weighted UniFrac β diversity, and MetaStat analysis) were evaluated.
RESULTS:
Dilution curve and species accumulation boxplot validated the quality of the samples. OTUs analysis of the 5 groups demonstrated that cervical bacterial genus consisted primarily of
CONCLUSIONS
The abundance of
Female
;
Humans
;
Microbiota
;
Papillomaviridae
;
Papillomavirus Infections
;
RNA, Ribosomal, 16S/genetics*
;
Uterine Cervical Neoplasms
;
Vaginal Smears
6.Infection and distribution characteristics of HPV of middle-aged and elderly women from a certain hospital in Guangxi Zhuang Autonomous Region from 2018 to 2020.
Xiao Ning WEI ; Xin Yu XU ; Shao Wei WANG
Chinese Journal of Preventive Medicine 2022;56(4):468-473
Objectives: To analyze the type and distribution characteristics of human papillomavirus (HPV) infection along with cervical cytology in middle-aged and elderly women in Guangxi and to provide a basis for the prevention and treatment of cervical cancer in elderly women. Methods: 21 subtypes of HPV and cervical cytology of women over 45-year-old visiting the First Affiliated Hospital of Guangxi Medical University from January 2019 to December 2020 were collected. They were divided into two groups by age, 45-64 years group and over 65 years group. The HPV, HR-HPV, and multiple HPV infection prevalence were analyzed, as well as HPV genotypes, the age distribution of HPV infection rate, and cervical cytology. Results: A total of 6 657 eligible women were included. 6 238 women were in the 45-64 years group, with a HPV prevalence about 20.86% (1 301), while 419 women were in the over 65 years group, with a HPV prevalence about 32.94% (138). The age-associated HPV and HR-HPV prevalence increased with the age, peaking at the age group of 70-74 years (P<0.001). The most prevalent genotype was HPV52, and the infection rate was 5.3% (353), followed by HPV16 and HPV 58, about 4.63% (308) and 3.08% (205) respectively. The majority cytology of HPV-positive middle-aged and elderly women was normal. 8.70% (88) of them were ASC-US, 6.52% (66) for HSIL, 4.55% (46) for LSIL, and 2.96% (30) for ASC-H, and 0.10% (1) for SCC. Compared to middle-aged women, elderly women had a lower negative cytology rate, 69.79% (67) vs. 77.95% (714), but a higher HSIL rate, 13.54% (13) vs. 5.79% (53) (P<0.05). Conclusions: HPV and HR-HPV prevalence of elderly women in a medical center of Guangxi are higher than those of middle-aged women. The most prevalent genotype is HPV16 in elderly women, followed by HPV52 and HPV58.
Aged
;
China/epidemiology*
;
Female
;
Hospitals
;
Human papillomavirus 16
;
Humans
;
Male
;
Middle Aged
;
Papillomaviridae/genetics*
;
Papillomavirus Infections/genetics*
;
Uterine Cervical Neoplasms
7.Association between CCND1 G870A polymorphism and radiotherapy response in high-risk human papillomavirus-related cervical cancer.
Shaoqiang LIANG ; Ning ZHANG ; Rong HUANG ; Jun JIANG ; Lusi CHEN ; Zhiqian LU ; Shaoen LI
Journal of Central South University(Medical Sciences) 2015;40(11):1205-1209
OBJECTIVE:
To investigate the correlation of cyclin D1 (CCND1) G870A single nucleotide polymorphism (SNP) with radiotherapy response in patients with high risk human papillomavirus (HR-HPV) related cervical cancer.
METHODS:
A total of 273 patients with cervical cancer, who were confirmed by histopathology and hybrid capture 2 (HC-2) assay and treated by radiotherapy, were enrolled for this study. The correlation of CCND1 G870A polymorphism with tumor response in patients was assessed.
RESULTS:
Compared with patients with AA genotype, the patients with GG genotype and AA genotype showed lower sensitivity to radio-therapy treatment (adjusted ORGA=2.69, 95% CI 1.28-5.67 and adjusted ORGG=3.28, 95% CI 1.47-7.29, respectively), an increase in risks of recurrence/metastasis (adjusted ORGA=2.52, 95% CI 1.12-5.63 and adjusted ORGG=3.95, 95% CI 1.68-9.26, respectively), and shorter recurrence/metastasis-free survival (PGA=0.010 and PGG=0.045).
CONCLUSION
G870A polymorphism is a frequent variation that could be used for evaluate the radio-sensitivity and prognosis for patients with HR-HPV related cervical cancer.
Cyclin D1
;
genetics
;
Female
;
Genotype
;
Humans
;
Papillomaviridae
;
Polymorphism, Single Nucleotide
;
Prognosis
;
Uterine Cervical Neoplasms
;
genetics
;
radiotherapy
;
virology
8.Human papillomavirus infection in men with condyloma acuminatum: Genotype analysis of 70 cases.
Qiang LI ; Yao-Wu GAO ; Hong-Qiang WANG ; Lei YU ; Tao JING ; Shen-Qian LI ; Pei-Tao WANG
National Journal of Andrology 2020;26(10):906-910
Objective:
To investigate the distribution of the gene subtypes of human papillomavirus (HPV) in male patients with condyloma acuminatum (CA) and analyze the characteristics of the gene subtypes.
METHODS:
We extracted genomic DNA of the HPV virus from the genital tissue of 70 male CA patients, detected the DNA subtypes of HPV using the PCR-reverse dot hybridization technique, and analyzed the rates of different subtypes identified and their characteristics of distribution in different age groups.
RESULTS:
The male HPV-positive patients were mainly infected at the age of 20-39 years, primarily with high- and low-risk mixed infection of various subtypes, which accounted for 61.54% in the 20- to 29-year-olds and 42.86% in the 30- to 39-year-olds. Among the 70 CA patients, 22 HPV subtypes were identified, the top five subtypes including HPV 11 (21.08%), HPV 6 (19.46%), HPV 42 (6.49%), HPV 59 (6.49%) and HPV 53 (5.95%); 20 infected with a single subtype (28.57%), 19 with two subtypes (27.14%) and 31 with three or more (44.29%); and 30 infected with a low-risk single subtype (42.86%) and 40 with both high- and low-risk multiple subtypes (57.14%).
CONCLUSIONS
Male patients with CA are mainly infected with HPV 11 and HPV 6, with a significantly higher rate of multi-subtype than single-subtype infection, and the multi-subtype patients chiefly with high- and low-risk mixed infection. Men aged 20-39 years old are most commonly affected by CA.
Adult
;
Condylomata Acuminata/virology*
;
DNA, Viral/genetics*
;
Genotype
;
Humans
;
Male
;
Papillomaviridae/genetics*
;
Papillomavirus Infections/virology*
;
Young Adult
9.Multichannel piezoelectric genesensor for the detection of human papilloma virus.
Jianghua WANG ; Weiling FU ; Minghua LIU ; Yingying WANG ; Qiang XUE ; Junfu HUANG ; Qianyong ZHU
Chinese Medical Journal 2002;115(3):439-442
OBJECTIVETo establish a method for rapid detection and sub-typing of human papilloma virus (HPV).
METHODSWe utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot.
RESULTSOf the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV.
CONCLUSIONOur results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.
Biosensing Techniques ; DNA, Viral ; analysis ; Humans ; Papillomaviridae ; classification ; genetics ; Papillomavirus Infections ; virology ; Polymerase Chain Reaction ; Tumor Virus Infections ; virology
10.Analysis of human papillomavirus type 61, 83 and 84 MY fragments and preparation of standard samples.
Bao-Chen WANG ; Xiao-Li WANG ; Feng-Xia XUE ; Hong-Tu LIU
Chinese Journal of Experimental and Clinical Virology 2010;24(2):107-109
OBJECTIVETo investigate the mutations in nucleotide and amino acid level in HPV61, 83 and 84 Shanxi isolates.
METHODSAmplified fragments of HPV61, 83 and 84 from human papillomavirus (Human papillomavirus, HPV) molecular epidemiologic survey of Shanxi Province using HPV consensus primers MY09/11 were cloned in pMD18-T vector, and the plasmids were sequenced, then nucleotide sequences and amino acid sequences were analyzed.
RESULTSHPV61 and HPV83 isolates were consistent with reference strains U31793 and AF151983 in nucleotide sequences; four mutations of nucleotide (C6760T, T6931C, T6951C and C6987A) were found in HPV84 isolate compared with reference strain AF293960, among which C6987A resulted in D441E and the amplified sensitivity of standard sample of HPV61 using primers MY09/11 was higher than that of HPV83 and 84.
CONCLUSIONHPV61 and HPV83 isolates were consistent with reference strains, four mutations of nucleotide and one mutation of amino acid were found in HPV84,the amplified sensitivity of standard sample of HPV61 using primers MY09/11 was the highest among those three isolates.
Base Sequence ; Genotype ; Humans ; Molecular Sequence Data ; Mutation ; Papillomaviridae ; genetics ; Papillomavirus Infections ; virology ; Sensitivity and Specificity ; Sequence Homology, Nucleic Acid