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Gagnon S Hankins C Tremblay C Pourreaux K Forest P Rouah F Coutlée F;Canadian Women's HIV Study Group François Coutlée 《Journal of medical virology》2005,75(2):213-221
The genomic polymorphism of high-risk human papillomavirus (HPV) for types other than 16 has not been extensively described. We describe here the genomic polymorphism of high-risk HPV type 31 in 79 women (62 HIV-seropositive, 17 HIV-seronegative) by PCR-sequencing of the long control region (LCR), E6 and E7. LCR polymorphism was generated by 25 (6.4%) single-nucleotide variations over 391 bases. Each variant compared to the prototype contained from 2 to 13 variations (mean of 9.4 +/- 3.3, median of 10). Considering the number of variation sites in each region of HPV genome, the LCR was more variable than E6 (13 over 496 nucleotide (nt), P=0.03) and E7 (9 over 296 nt, P=0.03). Non-synonymous nucleotide variations were found in 31 (75.6%) of 41 isolates and were observed at six positions in E6. Each of the 8 HPV-31 E7 variants contained from 2 to 5 mutations (mean of 4.29 +/- 1.11, median of 5) compared to the prototype. Three non-synonymous E6 and E7 variations were within cysteine arrays. The LCR prototype was significantly over-represented in Caucasian women (14 (25%) of 56) compared to women of African descent (0 (0%) of 15 women, P=0.03). Four (23.5%) of 17 women with persistent versus 6 (25.0%) of 24 women with transient infections were infected by the prototype (P=1.00). HPV-31 LCR was more polymorphic than oncogenes and was associated with ethnicity. 相似文献
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The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes. 相似文献
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Pande S Jain N Prusty BK Bhambhani S Gupta S Sharma R Batra S Das BC 《Journal of clinical microbiology》2008,46(3):1060-1066
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Shang Q Wang Y Fang Y Wei L Chen S Sun Y Li B Zhang F Gu H 《Journal of clinical microbiology》2011,49(7):2656-2663
Human papillomavirus type 16 (HPV 16) plays a cardinal role in the pathogenesis of cervical cancer. HPV 16 has intratypic variants which show different geographical distributions and different oncogenic potentials. To analyze the presence of sequence variations of HPV 16 variants in northeast China, 71 cervical carcinomas were identified by HPV typing. HPV 16-positive specimens were analyzed by PCR-directed sequencing in the E6, E7, and L1 genes and the LCR (long control region). The variation data were compared with those of neighboring districts. In this hospital-based study, HPV 16 was the most common type (73.24%). In HPV 16-positive specimens, 67.31% belonged to the European (E) lineage, while 32.69% were Asian (As) variants. The Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and northern American (NA) lineages were not detected. The most frequently observed variation sites were T178G (32.69%) in E6; A647G (34.62%), G666A (38.46%), and T846C (32.69%) in E7; C6826T (36.17%) and G7060A (61.70%) in L1; and G7521A (98.08%) in the LCR. The most prevalent amino acid variations were D25E in E6 and N29S in E7. In addition, 28 novel variations of HPV 16 were reported. Some covariations between different genes were obtained. In this study, HPV 16 variants belonged to the European lineage and the Asian lineage. Compared with neighboring districts, the distribution of HPV 16 variants in northeast China had a typical pattern. As the first report on HPV 16 variants in northeast China, it should be helpful for designing a HPV vaccine and HPV vaccination program in China. 相似文献
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Nucleotide sequence of human papillomavirus type 31: a cervical neoplasia-associated virus 总被引:18,自引:0,他引:18
The nucleotide sequence of human papillomavirus (HPV) 31 DNA (7912 bp) was determined and used to deduce the genomic organization of this cervical cancer-associated virus. Based on comparisons of the HPV 31 DNA sequence to other sequenced HPVs, HPV 31 is a typical papillomavirus most related to HPV 16 (70% identical nucleotides). The E6 and E7 open reading frames (ORF) of HPV 31 contain several potential DNA binding motifs (Cys-X-X-Cys), the locations of which are conserved in all HPVs. The E6 ORF also has the potential to code for an E6* protein. The E7 ORF of HPV31 encodes a polypeptide motif which appears to distinguish HPVs associated with cervical cancer, such as types 16, 18, 31, and 33, from HPVs found primarily in benign lesions, such as types 6 and 11. 相似文献
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Tainá Raiol Patrícia Soares Wyant Regina Maria Santos de Amorim Daniela Marreco Cerqueira Natália von Gal Milanezi Marcelo de Macedo Brígido Laura Sichero Cláudia Renata Fernandes Martins 《Journal of medical virology》2009,81(4):685-692
More than 100 HPV types have been described, 13 of which are classified as high‐risk due to their association with the development of cervical cancer. The intratype genomic diversity of HPV‐16 and ‐18 has been studied extensively, while little data have been generated for other less common high‐risk types. The present study explores the nucleotide variability and phylogeny of the high‐risk HPV‐31, ‐33, ‐35, ‐52, and ‐58, in samples from Central Brazil. For this purpose, the LCR and the E6 and L1 genes were sequenced. Several variants of these HPV types were detected, some of which have been detected in other parts of the world. Furthermore, new variants of all types examined were characterized in a total of 13 new variants. Based on the E6 and L1 sequences, variants were described comprising conservative and non‐conservative amino acid changes. For phylogenetic tree construction, samples characterized in this study were compared to others described and submitted to GenBank previously. The phylogenetic analysis of HPV‐31, ‐33, ‐35, and ‐58 isolates did not reveal ethnic or geographical clustering as observed previously for HPV‐16 and ‐18. HPV‐35 analysis showed a dichotomic branching characteristic of viral subtypes. Interestingly, four clusters relative to the analysis of HPV‐52 isolates were identified, two of which could be classified as Asian and European branches. The genomic characterization of HPV variants is crucial for understanding the intrinsic geographical relatedness and biological differences of these viruses and contributes further to studies on their infectivity and pathogenicity. J. Med. Virol. 81:685–692, 2009 © 2009 Wiley‐Liss, Inc. 相似文献
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The group of mucosal epithelia-infecting human papillomaviruses (HPV) can be subdivided in "low" and "high risk" HPV types. Both types induce benign neoplasia (condyloma), but only the infection with a "high risk" HPV type is causally associated with an increased risk of developing anogenital tumors. The oncogenic potential of high risk HPVs resides at least partially in the viral E6 protein. The E6 protein targets the cellular p53 protein for proteasome-dependent degradation, which is associated with the immortalizing and transforming functions of these viruses. Recently the E6-dependent proteasome-mediated destabilization of additional cellular proteins (E6TP1, c-myc, Bak, hMCM7, human scribble, E6AP, MAGI-1) has been described, but the cellular mechanisms controlling the viral E6 protein stability itself have been so far not analyzed. In this study, we transiently expressed the E6 genes of the high risk HPV type 16, the low risk HPV types 6a and 11, and the cutaneous epithelia-infecting HPV types 5 and 8 from a eucaryotic expression vector and compared the cellular steady-state levels of the expressed E6 proteins. We demonstrated that the high risk HPV 16 E6 protein possesses the lowest steady-state level in comparison to the low risk HPV type E6 proteins and the cutaneous epithelia-infecting HPV type E6 proteins. Inhibition of cellular proteasome-dependent protein degradation led to an increase in steady-state levels of high risk but not of low risk E6 proteins. Analysis of functionally deficient HPV 16 E6 proteins in p53 null- and p53 wild-type-expressing cell lines revealed that the cellular steady-state level of this protein is influenced neither by its p53- nor its E6AP-binding abilities. 相似文献
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Else EA Swoyer R Zhang Y Taddeo FJ Bryan JT Lawson J Van Hyfte I Roberts CC 《Journal of clinical microbiology》2011,49(5):1907-1912
Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays. 相似文献
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目的构建能表达L1E7融合蛋白的原核表达菌株,纯化蛋白,并观察其免疫效果。方法用PCR方法分别扩增出C末端部分缺失的HPV16L1基因和HPV16E7编码基因N端部分序列。将上述基因连接,构建融合基因L1ΔCE7N并将其插到原核表达载体pGEX-2T中进行融合蛋白表达纯化,然后观察其免疫效果。结果L1ΔCE7N融合基因测序结果表明,序列与设计相符,读码框架正确。将其插入原核表达质粒在大肠埃希菌中获得高效表达;经Wester-Blot鉴定在相对分子质量约85×103处有特异性表达带,与预期相符。用亲和层析和分子筛可纯化L1ΔCE7N融合蛋白,将其免疫C57BL/6小鼠,结果表明融合蛋白能诱发高滴度L1、E7抗体,并能保护小鼠免受TC-1肿瘤细胞的攻击。结论本实验在原核系统中高效表达并纯化了L1ΔCE7N融合蛋白,该蛋白可作为预防和治疗HPV16感染以及相关肿瘤的候选疫苗株。为研制HPV16预防治疗性疫苗探索一条经济、易普及的途径。 相似文献
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Raffaella Ghittoni Rosita Accardi Uzma Hasan Tarik Gheit Bakary Sylla Massimo Tommasino 《Virus genes》2010,40(1):1-13
More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous
or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group
of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which
represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is
the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk
mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV
persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk
mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host
immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation
of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing
a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the
findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV
types. 相似文献
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DNA-EIA to detect high and low risk HPV genotypes in cervical lesions with E6/E7 primer mediated multiplex PCR. 总被引:3,自引:2,他引:3
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C Clavel S Rihet M Masure C Chypre J C Boulanger C Quereux P Birembaut 《Journal of clinical pathology》1998,51(1):38-43
BACKGROUND: Oncogenicity of human papillomavirus (HPV) DNA in premalignant and malignant uterine cervical diseases is mainly induced by E6/E7 open reading frame (ORF). The presence of an oncogenic HPV DNA may be a diagnostic marker for the detection of cytologically negative smears. AIMS: To evaluate an original polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection and typing of oncogenic and non-oncogenic HPV types. METHODS: The test was an original multiplex labelled PCR-EIA for the detection and typing of oncogenic and non-oncogenic HPV using three consensus sequence primers within the oncogenic E6/E7 ORF. One primer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers could be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and non-oncogenic (6 and 11) HPV types in only two wells; then biotinylated oligoprobes were deposited in streptavidin-coated microplates. The PCR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (median age 32 years) at high risk for cervical cancer. RESULTS: HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) including 29 coinfections with oncogenic and non-oncogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade lesions: 95.8% of the cervical scrapes from patients with high grade lesions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytological examination of the cervical smears. Moreover, 60% of cervical scrapes exhibiting low grade lesions contained oncogenic HPV. CONCLUSIONS: This test is simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect simultaneously on a simple cervical scrape, two kinds of HPV--oncogenic and non-oncogenic--in just two microplate wells with non-isotopic oligoprobes. 相似文献