A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

Evaluation of different techniques for identification of human papillomavirus types of low prevalence

Human papillomaviruses (HPVs) have been recognized as etiologic factors in a variety of diseases. Due to the large number of HPV types, methods for HPV genotyping are difficult to standardize. Despite this fact, several methods exist, and some of them are available commercially. In this study, we evaluated the Roche Diagnostics linear array (LA) HPV genotyping assay, the Innogenetics INNO-LiPA (line probe assay [LiPA]), and two noncommercial reverse line blot (RLB) assays based on either primers GP5+ and GP6+ (GP) or newly designed broad-spectrum primers BSGP5+ and BSGP6+ (BS). The reliabilities of these assays were tested with a wide spectrum of HPV types less prevalent in cervical samples. This is the first study to compare the performance of the most widely used HPV genotyping methods with selected samples positive for low-prevalence HPV types. We focused on interassay agreement, both overall and type specific, in cases with single and/or multiple HPV infections. Interassay agreement was moderate in cases of single HPV infections and poor in cases of multiple HPV infections. The LA and the BS-based RLB assays found a higher rate of cases positive for multiple HPV types than LiPA and the GP-based RLB assay. The weakest capability in detecting multiple HPV infections was observed for LiPA. The use of only one assay in epidemiological and clinical studies might lead to biased conclusions. Therefore, a universally evaluated and agreed upon HPV typing assay or a combination of current assays is needed for possible clinical applications, and knowledge of their limitations is advised.     

Multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers

With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC₅₀s of <16 nM against genotype 1 and >100 nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC₅₀ to levels comparable to those for genotype 1a, but did not considerably alter the EC₅₀ of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a.     

The carcinogenicity of human papillomavirus types reflects viral evolution

Persistent infections with carcinogenic human papillomaviruses (HPV) cause virtually all cervical cancers. Cervical HPV types (n > 40) also represent the most common sexually transmitted agents, and most infections clear in 1-2 years. The risks of persistence and neoplastic progression to cancer and its histologic precursor, cervical intraepithelial neoplasia grade 3 (CIN3), differ markedly by HPV type. To study type-specific HPV natural history, we conducted a 10,000-woman, population-based prospective study of HPV infections and CIN3/cancer in Guanacaste, Costa Rica. By studying large numbers of women, we wished to separate viral persistence from neoplastic progression. We observed a strong concordance of newly-revised HPV evolutionary groupings with the separate risks of persistence and progression to CIN3/cancer. HPV16 was uniquely likely both to persist and to cause neoplastic progression when it persisted, making it a remarkably powerful human carcinogen that merits separate clinical consideration. Specifically, 19.9% of HPV16-infected women were diagnosed with CIN3/cancer at enrollment or during the five-year follow-up. Other carcinogenic types, many related to HPV16, were not particularly persistent but could cause neoplastic progression, at lower rates than HPV16, if they did persist. Some low-risk types were persistent but, nevertheless, virtually never caused CIN3. Therefore, carcinogenicity is not strictly a function of persistence. Separately, we noted that the carcinogenic HPV types code for an E5 protein, whereas most low-risk types either lack a definable homologous E5 ORF and/or a translation start codon for E5. These results present several clear clues and research directions in our ongoing efforts to understand HPV carcinogenesis.     

Bead-based multiplex genotyping of 58 cutaneous human papillomavirus types

Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested "hanging-droplet" FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.     

Diversity of cutaneous human papillomavirus types in individuals with and without skin lesion.

BACKGROUND: Human papillomavirus (HPV) is ubiquitous on the skin of normal and immunosuppressed hosts. OBJECTIVE: We describe the diversity of HPV types in skin specimens using PCR-sequencing directly and after cloning with FAP59/64 or HVP2/B5 primers. STUDY DESIGN: Cross-sectional analysis of skin swabs. RESULTS: Seventy-five (92.6%) of 81 subjects provided samples that could be analysed with PCR (34 healthy controls <50 years old, 13 healthy controls > or =50 years old, 12 with actinic keratosis (AK), 8 with squamous cell carcinoma (SCC) and 8 renal transplant recipients). HPV DNA was detected more frequently with FAP59/64 (68/75, 91%) than with HVP2/B5 (9/75, 12%) (p<0.001). Agreement of typing results using FAP59/64 primers with both sequencing strategies was fair (mean kappa 0.56+/-0.19, 95% CI: 0.46-0.65). HPV species 1 and 2 of the beta-papillomavirus genus were associated with the presence of AK (OR=24.8, 95% CI: 2.3-262.6). A greater number of HPV types per sample was found in individuals with AK or SCC (p=0.046) or AK alone (p=0.02), than in healthy participants. CONCLUSION: HPV infection on the skin is best evaluated with a combination of primers and sequencing strategies. Novel putative types were frequently detected in SCC. Skin lesions have a greater number of HPV types than normal skin.     

Yeast coexpression of human papillomavirus types 6 and 16 capsid proteins

The L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29-1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types.     

Deceiving high-grade cervical dysplasias identified as human papillomavirus non-16 and non-18 types by Invader human papillomavirus assays

High-grade cervical intraepithelial lesions (HGCINs) are easily diagnosed by established histologic criteria. However, we encountered problematic cases that are difficult to diagnose because features intermediate between dysplasia and metaplasia are present. p16 and Ki-67 immunostains proved HGCIN in these difficult and unusual cases. Because these are unusual cases of cervical dysplasia, we decided to type the human papillomavirus (HPV) using the Invader HPV test with analyte-specific reagents developed by Third Wave Technologies (Madison, WI, USA) (a new HPV screening assay applicable to tissue and amenable to rapid, sensitive, and specific detection of 14 high- to intermediate-risk HPV types) and a panel of immunostains. Results of these difficult cases are compared with classic HGCIN cases. We searched our pathology files over a period of 16 months for high-grade squamous intraepithelial lesion, cervical intraepithelial neoplasia II, cervical intraepithelial neoplasia III, and p16. To identify cases of difficult HGCIN with features intermediate between dysplasia and metaplasia, we reviewed all surgical cases of HGCIN that required p16 and Ki-67 diagnosis confirmation. Cases of interest were also stained with ProExC. Human papillomavirus screening and HPV 16/18 typing were performed by the Invader assays as described previously. Ten cases of classic HGCIN were easily diagnosed by hypercellularity, significant atypia, mitotic figures, and diffuse staining by p16, Ki67 and ProExC. The Invader assay identified HPV 16 (A9 positive/HPV16 positive) in 7 of 10 cases; the 3 others were A7 positive/not HPV18 (1) and A9 positive/not HPV16 (2). Eight cases of difficult HGCIN were identified. These showed only mild-to-moderate cellularity, a lack of significant atypia, absent-to-rare mitotic figures, and diffuse staining by p16, Ki-67, and ProExC. Human papillomavirus DNA was detected in 5 of 8 cases: only 1 was A9 positive/HPV16 positive, 1 was A5/A6 positive, 1 was A7 positive/not HPV18, and 2 were A9 positive/not HPV16. Three remaining cases demonstrated sufficient DNA to be analyzed by the Invader assay, but results were negative. This is a poorly recognized unusual group of cervical HGCIN with features intermediate between dysplasia and metaplasia that is easily confused by histologic examination. Immunostains prove the high-grade nature of these lesions, and Invader assay demonstrates association with HPV types other than 16/18 (ie, other HPV types detected by Invader assay). In this study, we present an unusual group of cases of high-grade dysplasia, not recognized by hematoxylin and eosin but identified by Ki67 and P16. It is very important to emphasize that this unusual group of high-grade dysplasias is associated with high-risk HPV but with types other than 16/18.     

Integration of human papillomavirus types 16 and 18 in cervical adenocarcinoma.

AIMS: To determine which type of human papillomavirus (HPV) is associated with cervical adenocarcinoma and whether the virus was integrated or episomal in two continents. METHODS: Biopsy specimens from the UK (n = 16) and South Africa (n = 22) were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, 33, and 35 on archival biopsy specimens using digoxigenin labelled probes. RESULTS: A total of 20 adenocarcinomas (53%) from both groups contained HPV DNA. In the UK group, seven and four cases contained HPV 18 (44%) and 16 (25%) respectively. In the South African group, nine cases contained HPV 18 (41%) while HPV DNA was not detectable in the other 13 cases. Hence HPV 18 was present in 80% of HPV positive adenocarcinomas. CONCLUSIONS: The HPV 16 or 18 genome was integrated in all viral positive cases. In two cases HPV 18 was also present in an episomal form. These data indicate that HPV integration is common to cervical adenocarcinoma in two continents by the same methodology. The lower prevalence of HPV 18 detection in the South African group may have been due to the presence of other or unsequenced HPV types.     

Mapping of human serum-reactive epitopes in virus-like particles of human papillomavirus types 16 and 11

Most human antibodies against HPV16 can be blocked by the monoclonal antibody H16.V5. To investigate whether H16.V5 and human sera recognize similar epitopes, hybrid capsids containing different parts of HPV16 and HPV11 were evaluated for reactivity with human sera. The antibody responses among HPV 16-/HPV11+sera to HPV11 and to hybrid capsids containing the HPV11 C-terminus were strongly correlated. The antibody responses among HPV 16+/HPV11-sera to HPV16 and to a hybrid containing the HPV16 C-terminus were correlated and there was also reactivity with a hybrid containing the H16.V5 epitope in the HPV11 backbone. Several HPV16-/11- children's sera were reactive with hybrid capsids, implying that a native capsid structure is essential for serological specificity. For both HPV16 and HPV11, the major serologic reactivity was directed toward the C-terminal part of the protein and the H16.V5 binding site appeared to be a major serologically reactive epitope of HPV16.     

人乳头状瘤病毒16、18型变异及相关肿瘤

肿瘤的形成可由多种病毒引起[1],其一就是人类乳头状瘤病毒(Human papillomavirus,HPV).HPV是1949年从普通疣体浸出液中首次被观察到,1995年有研究证实HPV感染为宫颈癌的致病病原体.研究显示:HPV16、18能促使人类鳞状上皮的增生.目前HPV16、18变异体的致瘤性受到各国学者的广泛关注.     

Use of multiple displacement amplification in the investigation of human papillomavirus physical status

BACKGROUND AND AIMS: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities. METHODS: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation. RESULTS: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples. CONCLUSIONS: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.     

Distribution of human papillomavirus types in the anogenital tract of females and males

Human papillomavirus (HPV) infection is the most common sexually transmitted infection in both men and women, but there are limited data comparing the prevalence of HPV infection between genders and in different anogenital sites. This cross‐sectional analysis describes the distribution of HPV types in the genital tract of 3,410 consecutive females and 1,033 males undergoing voluntary screening for HPV and referred to a single institution. The relationship between specific HPV types and the presence of anogenital lesions was examined. In both females and males, the overall prevalence of HPV infection was about 40%. A wide variety of HPV types was identified, but the prevalence of different types was remarkably similar in the two genders, even when considering different anatomical sites. HPV‐6 was the most frequent (prevalence 13%) type in all anogenital sites in men followed by HPV‐16 (7%), while HPV‐16 was the most common type in women (about 6%), either in the cervix, vagina, or vulva, followed by HPV‐6. In addition to HPV‐16, HPV‐58, HPV‐33, HPV‐31, and HPV‐56 were the carcinogenic types detected most commonly and were significantly associated with high‐grade squamous intraepithelial cervical lesions, while HPV‐53 and HPV‐66 were the most common among possibly carcinogenic types. In both genders, anogenital warts were associated with HPV‐6 and HPV‐11 infection, and, less frequently, with other types, like HPV‐54, HPV‐62, and HPV‐66. These results show that genital HPV infection involves numerous HPV types, which have similar distribution patterns in females and males and in different anogenital anatomical sites. J. Med. Virol. 82:1424–1430, 2010. © 2010 Wiley‐Liss, Inc.     

Simultaneous detection and typing of human papillomavirus in cervical biopsies using PCR-reverse hybridization

INTRODUCTION: Genotyping of Human papillomavirus (HPV) is an important step in the clinical evaluation of the oncogenic risk associated with HPV infection of cervical mucosa. The purpose of this work was to develop a fast PCR-reverse-hybridization assay (PCR-RH) for the simultaneous detection and genotyping of anogenital HPVs. METHODS: HPV DNA from cervical biopsies was amplified by consensus primer-PCR. Digoxigenin-labeled PCR products were hybridized to type-specific probes anchored to the surface of plastic microwells and revealed by an ELISA system. RESULTS: The method was tested on 115 clinical samples (81 koilocytic atypias, 11 CIN1, 10 CIN2, 12 CIN3 and 1 squamous carcinoma). HPV DNA was found in 56.7% koilocytic atypias, in 90.9% of CIN1 and in 100% of CIN2 and higher-grade lesions. Thus, PCR-RH is sensitive, rapid, easy-to-perform and readily applicable to the routine analysis of a large number of samples.     

Detection of human papillomavirus types 45 and 51 by type-specific polymerase chain reaction

Human papillomavirus (HPV) types 45 and 51 are both considered as high risk types for the development of human cervical cancer. To optimize the detection of these two types in clinical samples, HPV-45 and HPV-51 specific primers were designed to amplify respectively a 141bp and a 266bp fragment from the L1 gene by polymerase chain reaction (PCR). The sensitivity and the specificity of these two PCR reactions were determined using varying amounts of HPV DNA containing plasmids and negative and positive controls. Overall, the sensitivity for the HPV-45 plasmid DNA is 10fg, while for HPV-51 the sensitivity is 1fg. This is equivalent to approximately 100 and 10 HPV genome copies per PCR reaction, respectively.     

Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia

Some human papillomavirus (HPV) types, such as HPV 16, are clearly associated with cervical dysplasia; however, the role played by other HPV types occasionally found in dysplasia is less certain. In addition, most methods used to detect HPV in clinical specimens cannot easily distinguish among more than two or three HPV types in a single specimen. Therefore, the significance of infection with multiple HPV types is not known. To address this question, we analyzed cervicovaginal lavage specimens from three cohorts of women for HPV DNA using a PCR/reverse blot assay system that permits the detection and partial quantitation of 26 genital HPV types. As expected, 94.1% of women who had dysplasia (n = 34) and 71.4% of women who had atypical squamous cells of uncertain significance (ASCUS) (n = 21) on cytology had HPV DNA detected compared to 54.5% of age matched women with normal cytology. HPV 16 DNA was detected in 35% of dysplasia patients compared to 9% of cytologic normals (P = 0.0044). Dysplasia patients had a mean of 3.29 (range 0-10) different HPV types detected compared to 1.04 (range 0-7) HPV types among those with normal cytology (P < 0.0001). These data support a possible role for multiple HPV types in the development or progression of cervical dysplasia.