Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3′ ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3′ ends to generate the primers GP5+ and GP6+, respectively. Compared with the GP5/6 PCR, GP5+/6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10- to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5+/6+ based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39% (GP5/6 PCR) to 43% (GP5+/6+PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5+/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.

The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR.

The E6 oncoprotein derived from tumour-associated human papillomaviruses (HPVs) binds to and induces the degradation of the cellular tumour-suppressor protein p53 A common polymorphism that occurs in the p53 amino-acid sequence results in the presence of either a proline or an arginine at position 72 The effect of this polymorphism on the susceptibility of p53 to E6-mediated degradation has been investigated and the arginine form of p53 was found to be significantly more susceptible than the proline form Moreover, allelic analysis of patients with HPV-associated tumours revealed a striking overrepresentation of homozygous arginine-72 p53 compared with the normal population, which indicated that individuals homozygous for arginine 72 are about seven times more susceptible to HPV-associated tumorigenesis than heterozygotes The arginine-encoding allele therefore represents a significant risk factor in the development of HPV-associated cancers

Role of a p53 polymorphism in the development of human papillomavirus-associated cancer.

The presence of genital human papillomavirus (HPV) was determined at cervical and vulvar sites using two methods, the Food and Drug Administration-approved ViraPap test and polymerase chain reaction (PCR) DNA amplification technology, in 467 women presenting to a university health service for a routine annual gynecologic examination. The PCR system afforded the sensitive detection of a broad spectrum of genital HPV types. Using PCR, we found that 46% of the study population was infected with HPV; the ViraPap test showed a prevalence of 11% infected. PCR analyses demonstrated that 69% of the HPV-positive women were infected at both genital sites. Subsequent HPV-type determination showed that 33% of the study population had HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, or other previously isolated types, and 13% had yet unidentified types. Almost all (92%) of the women diagnosed by Papanicolaou smear with condylomatous atypia or dysplasia (n = 12) were HPV positive. The PCR method proved to be an informative and rapid way to detect HPV in large numbers of clinical samples. Our results demonstrate that genital HPV infection is common among sexually active young women.

https://jamanetwork.com/HttpHandlers/ArticlePdfHandler.ashx?journal=JAMA&articleId=384835&pdfFileName=jama_265_4_027.pdf

Genital human papillomavirus infection in female university students as determined by a PCR-based method.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1,354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5+/6+ primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

/pdf/development-and-clinical-evaluation-of-a-highly-sensitive-3qkgl2s9y6.pdf

Development and Clinical Evaluation of a Highly Sensitive PCR-Reverse Hybridization Line Probe Assay for Detection and Identification of Anogenital Human Papillomavirus

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.

GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes

A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.

The Use of General Primers in the Polymerase Chain Reaction Permits the Detection of a Broad Spectrum of Human Papillomavirus Genotypes

At present human papillomaviruses (HPVs) are linked to up to 10% of the worldwide cancer burden, mainly due to their involvement in anogenital cancer. Accumulating experimental data on cervical squamous cell carcinoma strongly support the assumption that certain HPV types have carcinogenic potential and are causally related to cervical cancer (zur Hausen 1991). H PV-16 and HPV-18, the major types associated with cervical cancer, exhibit transforming and immortalizing functions in primary rodent cells and human keratinocytes, respectively (Matlashewski et al. 1987; Pecoraro et al. 1989; Pirisi et al. 1987), and the viral E6 and E7 open reading frames (ORFs) have been found to encode the HPV-transforming proteins (Barbosa and Schlegel 1989; Hawley-Nelson et al. 1989; Munger et al. 1989a). The HPV-16 and HPV-18 E6 and E7 proteins can form complexes with the tumor suppressor gene products p53 and pRB, respectively (Werness et al. 1990; Munger et al. 1989b), which at least in part may explain at what level these HPV types interfere with cell cycle control mechanisms. However, viral E6–E7 functions are not sufficient for the development of malignant growth, and the additional modification of host cell genes by chemical factors has been considered to be important as well (zur Hausen 1982, 1989). Owing to this hypothesis, interest has been provoked for a role of HPV in the pathogenesis of carcinomas within the upper digestive and respiratory tracts since these tumors are known to be related to chemical factors such as tobacco and alcohol. Worldwide, squamous cell carcinoma of the ororespiratory tract, especially that of the lung, is the major form of cancer in humans. Therefore, efforts to study the relationship of specific HPV types with carcinomas of this tract are justified.

Papillomaviruses and Cancer of the Upper Digestive and Respiratory Tracts

Recent developments in tumor virology have shown that specific human papillomaviruses (HPV) play an important role in the development of cervical cancer (Gissmann and Schneider 1987; Zur Hausen 1989). In general, HPV 6 and 11 have been associated with benign cervical lesions (Gissmann et al. 1983; McCane et al. 1985; Lorincz et al. 1986a) while HPV 16, 18 and to a lesser extent HPV 31, 33, and 35 have been found mainly in mild and severe dysplasia and cervical carcinoma (Durst et al. 1983; Boshart et al. 1984; Beaudenon et al. 1986; Lorincz et al. 1986b).

Detection of Genital Human Papilloma Virus Infections by the Polymerase Chain Reaction

Certain human papillomavirus (HPV) genotypes are considered to play a crucial. role in the development of cervical. neoplasia, in particular HPV 16. However there are several. indications that additional. events besides HPV infection are involved in the pathogenesis of cervical. cancer1. A hint that failure of the immune response might play a role is given by the reduced expression of major histocompatibility complex class I (MHC-I), frequently observed in cervical. Carcinomas2,3. This could facilitate escape of neoplastic cells from killing by cytotoxic T-cells (CTLs), since cell surface expression of MHC-I molecules is necessary for recognition and subsequent killing of antigen bearing cells by CTLs4. It is tempting to speculate on a role of HPV in MHC-I down-regulation, in analogy with other viruses5. In a previous study we focused on the E7 gene of HPV 16, since this gene is consistently expressed in cervical. carcinomas6. The HPV 16-E7 protein shows functional. homology with the Adenovirus El A gene product7, which is responsible for MHC-I down-regulation8. However, the observed reduction of class I expression was not found in all HPV 16-E7 expressing carcinomas3, indicating no direct effect of E7 on down-regulation of MHC-I expression.

A. J. C. van den Brule

Papers

The Use of General Primers in the Polymerase Chain Reaction Permits the Detection of a Broad Spectrum of Human Papillomavirus Genotypes

Papillomaviruses and Cancer of the Upper Digestive and Respiratory Tracts

Detection of Genital Human Papilloma Virus Infections by the Polymerase Chain Reaction

Analysis of MHC Class I Expression in HPV 16 Positive Cervical Carcinomas, in Relation to C-MYC Overexpression