Genetic diversity and phylogenetic analysis of HPV 16 & 18 variants isolated from cervical specimens of women in Saudi Arabia

Human papillomavirus (HPV) are well known to be associated with the development of cervical cancer. HPV16 and HPV 18 are known as high-risk types and reported to be predominantly associated with cervical cancer. The prevalence and genetic diversity of HPV have been well documented globally but, in the Kingdom of Saudi Arabia, data on HPV genetic diversity are lacking. In this study, we have analyzed the genetic diversity of both HPV16 and HPV18 based on their L1 gene sequence because L1 gene is a major capsid protein gene and has been utilized to develop a prophylactic vaccine. In January 2011–2012, a total of forty samples from cervical specimens of women in Saudi Arabia were collected. The association of HPV16, HPV18 was detected by polymerase chain reaction, sequenced and submitted to GenBank. The sequences identity matrix and the phylogenetic relationship were analyzed with selected HPVs. The highest sequence identity (99.5%) for HPV16 and (99.3%) for HPV was observed with selected HPVs. The phylogenetic analysis results showed that HPVs from Saudi Arabia formed a closed cluster with African, Asian, East Asian as well as American HPVs distributed into multiple linages from various geographical locations. The results provided the valuable information about genetic diversity, but there is an urgent need to generate full genome sequence information which will provide a clearer picture of the genetic diversity and evolution of HPVs in Saudi Arabia. In conclusion, the generated data will be highly beneficial for developing molecular diagnostic tools, analyzing and correlating the epidemiological data to determine the risk of cervical cancer and finally to develop a vaccine for Saudi Arabian population.     

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer.     

Crystal structures of four types of human papillomavirus L1 capsid proteins: understanding the specificity of neutralizing monoclonal antibodies

Human papillomaviruses (HPVs) are known etiologic agents of cervical cancer. Vaccines that contain virus-like particles (VLPs) made of L1 capsid protein from several high risk HPV types have proven to be effective against HPV infections. Raising high levels of neutralizing antibodies against each HPV type is believed to be the primary mechanism of protection, gained by vaccination. Antibodies elicited by a particular HPV type are highly specific to that particular HPV type and show little or no cross-reactivity between HPV types. With an intention to understand the interplay between the L1 structure of different HPV types and the type specificity of neutralizing antibodies, we have prepared the L1 pentamers of four different HPV types, HPV11, HPV16, HPV18, and HPV35. The pentamers only bind the type-specific neutralizing monoclonal antibodies (NmAbs) that are raised against the VLP of the corresponding HPV type, implying that the surface loop structures of the pentamers from each type are distinctive and functionally active as VLPs in terms of antibody binding. We have determined the crystal structures of all four L1 pentamers, and their comparisons revealed characteristic conformational differences of the surface loops that contain the known epitopes for the NmAbs. On the basis of these distinct surface loop structures, we have provided a molecular explanation for the type specificity of NmAbs against HPV infection.     

HPV感染与宫颈癌预防的研究进展

宫颈癌在全球范围内依然是严重威胁妇女健康的常见恶性肿瘤之一。流行病学调查显示,高危型HPV持续感染是导致宫颈癌发病的主要原因,并且HPV感染具有显著的特点,因此,预防HPV感染是防治宫颈癌的主要途径。已明确性行为是促进HPV感染最重要的辅助因子,个体免疫力低下及年龄因素亦是促进HPV感染的重要因素。研究显示,在全球逐渐开展的预防措施包括对常见的高风险HPV类型进行预防性疫苗接种及包括HPV检测在内的宫颈癌筛查项目的实施已经在降低宫颈癌的发病率方面起到了很重要的作用。近些年来,人们越来越重视HPV检测在宫颈癌筛查程序中的应用。由于HPV的感染率具有显著的地域性差异,我们需要针对各地区的特点以完善相应的宫颈癌筛查程序,从而为宫颈癌防治工作的开展提供重要依据。     

Papillomviren und Krebs. Wie aus einem Anfangsverdacht ein Impfstoff wurde

Since a long time epidemiologic studies suggested a sexually transmitted infectious agent as cause of cervical cancer. Human papillomaviruses (HPV) were considered as causative agents since members of the papillomavirus family can induce tumors in animals, and genital infections with papillomaviruses in man are common. Several discoveries ranging from the identification of the plurality of HPVs, the regular finding of genomes of certain, so called high‐risk HPV types within tumor biopsies, insights into the transforming mechanism and informative epidemiological surveys finally led to the conclusion within the scientific community of a causative link between these viruses and cervical cancer as well as other human malignancies. This awareness triggered the development of HPV‐specific vaccines as means of cancer prevention.     

Carrageenan is a potent inhibitor of papillomavirus infection

Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate-independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs.     

Skin cancer and virus]

Human papillomaviruses (HPV) generally associated with benign skin and anogenital warts. Because several of skin cancers were found to contain HPV-DNA, it has been speculated that certain types of HPV could be specifically associated with cancers. Although HPV-DNAs are not isolated from most of skin cancers, they are often isolated from penile cancers, vulval cancers and anogenital Bowen's diseases. Patients with epidermodysplasia verruciformis start to suffer from disseminated incurable warts during their childhood, and some of these benign lesions often convert to skin cancer in adulthood. Although the disease is very rare, HPV may also play a role in malignant transformation in epidermodysplasia verruciformis. More than 60 types of HPVs distinguished by molecular hybridization techniques. The type of HPV determines the clinical picture of wart and natural fate of HPV-associated lesion. There are two groups of HPVs, which are benign types and malignant types. Viral DNA of malignant type of HPV transforms human keratinocytes in vitro, and the transforming activity has been mapped to the E6 and E7 genes.     

PKN binds and phosphorylates human papillomavirus E6 oncoprotein

The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two viral oncoproteins E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary epithelial cells has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.     

Prevention of cervical cancer through papillomavirus vaccination

A subset of human papillomaviruses (HPVs) promote anogenital malignancy, including cervical cancer, and prevention and treatment strategies that reflect the causal role of HPV are being developed. Vaccines based on HPV virus-like particles induce genotype-specific virus-neutralizing antibody and prevent infection with HPV. Persistent papillomavirus infection is required for the development of papillomavirus-associated cancer and, therefore, therapeutic vaccines are being developed to eliminate established papillomavirus infection. Such vaccines test principles for the growing field of tumour-antigen-specific immunotherapy. This article reviews progress in the field and draws conclusions for the development of future prophylactic and therapeutic viral vaccines.     

Economic Burden of Human Papillomavirus-Related Diseases in Italy

Introduction

Human papilloma virus (HPV) genotypes 6, 11, 16, and 18 impose a substantial burden of direct costs on the Italian National Health Service that has never been quantified fully. The main objective of the present study was to address this gap: (1) by estimating the total direct medical costs associated with nine major HPV-related diseases, namely invasive cervical cancer, cervical dysplasia, cancer of the vulva, vagina, anus, penis, and head and neck, anogenital warts, and recurrent respiratory papillomatosis, and (2) by providing an aggregate measure of the total economic burden attributable to HPV 6, 11, 16, and 18 infection.

Methods

For each of the nine conditions, we used available Italian secondary data to estimate the lifetime cost per case, the number of incident cases of each disease, the total economic burden, and the relative prevalence of HPV types 6, 11, 16, and 18, in order to estimate the aggregate fraction of the total economic burden attributable to HPV infection.

Results

The total direct costs (expressed in 2011 Euro) associated with the annual incident cases of the nine HPV-related conditions included in the analysis were estimated to be €528.6 million, with a plausible range of €480.1–686.2 million. The fraction attributable to HPV 6, 11, 16, and 18 was €291.0 (range €274.5–315.7 million), accounting for approximately 55% of the total annual burden of HPV-related disease in Italy.

Conclusions

The results provided a plausible estimate of the significant economic burden imposed by the most prevalent HPV-related diseases on the Italian welfare system. The fraction of the total direct lifetime costs attributable to HPV 6, 11, 16, and 18 infections, and the economic burden of noncervical HPV-related diseases carried by men, were found to be cost drivers relevant to the making of informed decisions about future investments in programmes of HPV prevention.     

Anogenital warts contain several distinct species of human papillomavirus.

Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2. In 12 tissue samples lacking sequence homology to either HPV-1 or HPV-2 under stringent conditions, HPV-related sequences were detected when the hybridization was performed under less stringent conditions, indicating that an HPV distinct from both HPV-1 and HPV-2 is also associated with these lesions. This anogenital HPV also appeared to be distinct from the other characterized types of HPV. These data indicate that at least three HPVs are associated with anogenital wart disease.     

Frequency of human papillomavirus types 16, 18, 31, and 33 and sites of cervical lesions in gynecological patients from Recife, Brazil

Human papilloma virus (HPV) is a well-established cause of cervical cancer. While many studies have been performed so far on HPV viral biology, mode of infection and prevention measures, scanty information is available on lesion sites of infected women and the incidence of viral types at specific locations. We looked for a possible relationship between the most common viral types (HPVs 16, 18, 31, 33) found in Recife, PE, Brazil, and lesion sites. We examined 396 HPV-positive women at the Gynecological Unit of the IMIP at Recife; 288 women were positive for HPV 16, 18, 31, or 33, present as a single-virus type or as co-infection. HPV 16 was the most frequent virus type found in the vulva, vagina, uterine cervix-vagina, and uterine cervix. HPV 31 was the second prevalent virus type in vulva, vagina, uterine cervix-vagina, uterine cervix, and mole. HPVs 18 and 33 were present with similar frequencies in the mole-vulva region. Among the co-infections, HPV 16/18 and HPV16/31 were the most frequent in our study group, followed by HPV 16/33.     

Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins

Low-risk type human papillomavirus (HPV) 6 and 11 infection causes recurrent respiratory papillomatosis (RRP) and genital warts. RRP is the most common benign tumor of the larynx in children with frequent relapses. Repeated surgeries are often needed to improve vocal function and prevent life-threatening respiratory obstruction. Currently, there are no effective treatments available to completely eliminate these diseases, largely due to limited knowledge regarding their viral molecular pathogenesis. HPV E6 proteins contribute to cell immortalization by interacting with a variety of cellular proteins, which have been well studied for the high-risk type HPVs related to cancer progression. However, the functions of low-risk HPV E6 proteins are largely unknown. In this study, we report GST-pulldown coupled mass spectrometry analysis with low-risk HPV E6 proteins that identified sterile alpha motif domain containing 9 (SAMD9) as a novel interacting partner. We then confirmed the interaction between HPV-E6 and SAMD9 using co-immunoprecipitation, proximity ligation assay, and confocal immunofluorescence staining. The SAMD9 gene is down-regulated in a variety of neoplasms and deleteriously mutated in normophosphatemic familial tumoral calcinosis. Interestingly, SAMD9 also has antiviral functions against poxvirus. Our study adds to the limited knowledge of the molecular properties of low-risk HPVs and describes new potential functions for the low-risk HPV E6 protein.     

PCR based detection of HPV 16 and 18 genotypes in normal oral mucosa of tobacco users and non-users

There is increasing evidence of a causal association between human papillomavirus (HPV) and oral squamous cell carcinoma (OSCC). Several studies have shown that HPV is associated with increased risk of oral cancer independent of exposure to tobacco and alcohol. The association is valid for HPVs 16 and 18, which generally are considered high risk types, because they have been detected in oral dysplastic lesions and cancers. We determined the baseline prevalence of HPVs 16 and 18 in normal oral mucosa of individuals with and without tobacco habit. PCR was used for DNA collected by oral smears to detect HPV 16/18 DNA in normal oral mucosa of 60 healthy individuals who were assigned to two groups of 30 subjects each. One group had a tobacco habit, the other did not. The tobacco user group comprised individuals who were tobacco chewers only. Sixty-five percent of individuals were positive for HPV 16/18 DNA, but HPV 16/18 positivity was less in individuals with tobacco habit than in those without tobacco habit. No significant association was found between the presence of HPVs and gender, age or duration of chewing habit, or between groups with and without a tobacco habit. We propose that HPVs16 and 18 commonly are present in normal oral mucosa and emphasize the importance of distinguishing clinical, subclinical and latent HPV infections when investigating HPVs and OSCC.     

Potential impact of a 9-valent HPV vaccine in HPV-related cervical disease in 4 emerging countries (Brazil,Mexico, India and China)

BackgroundWe estimated the potential impact of an investigational 9-valent human papillomavirus (HPV) vaccine (HPVs 6/11/16/18/31/33/45/52/58) in HPV-related cervical disease in Brazil, Mexico, India and China, to help to formulate recommendations on cervical cancer prevention and control.MethodsEstimations for invasive cervical cancer (ICC) were based on an international study including 1356 HPV-positive cases for the four countries altogether, and estimations for precancerous cervical lesions were extracted from a published meta-analysis including 6 025 HPV-positive women from the four mentioned countries. Globocan 2012 and 2012 World Population Prospects were used to estimate current and future projections of new ICC cases.ResultsCombined proportions of the 9 HPV types in ICC were 88.6% (95%CI: 85.2–91.3) in Brazil, 85.7% (82.3–88.8) in Mexico, 92.2% (87.9–95.3) in India and 97.3% (93.9–99.1) in China. The additional HPV 31/33/45/52/58 proportions were 18.8% (15.3–22.7) in Brazil, 17.6% (14.2–21.2) in Mexico, 11.3% (7.5–16.1) in India and 11.9% (7.5–17.2) in China. HPV6 and 11 single types were not identified in any of the samples.Proportion of the individual 7 high risk HPV types included in the vaccine varied by cytological and histological grades of HPV-positive precancerous cervical lesions. HPV 16 was the dominant type in all lesions, with contributions in low grade lesions ranging from 16.6%(14.3–19.2) in Mexico to 39.8% (30.0–50.2) in India, and contributions in high grade lesions ranging from 43.8% (36.3–51.4) in Mexico to 64.1% (60.6–67.5) in Brazil. After HPV 16, variations in other majors HPV types were observed by country, with an under representation of HPV 18 and 45 compared to ICC.ConclusionThe addition of HPVs 31/33/45/52/58 to HPV types included in current vaccines could increase the ICC preventable fraction in a range of 12 to 19% across the four countries, accounting the 9-types altogether 90% of ICC cases. Assuming the same degree of efficacy of current vaccines, the implementation of the 9-valent HPV vaccine in Brazil, Mexico, India and China would substantially impact on the reduction of the world cervical cancer burden.     

Persistence of human papillomavirus infection: keys to malignant progression

Human papillomaviruses (HPVs) are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Although prophylactic vaccines have been developed that target high-risk HPV types, there is a continuing need to understand better the virus-host interactions that underlie persistent benign infection and progression to cancer. In this review we summarize the molecular events that facilitate the differentiation-dependent HPV life cycle, how the life cycle is organized to facilitate virus persistence, and how the activities of HPV regulatory proteins result in malignancy.