Characterization of a New Type of Human Papillomavirus That Causes Skin Warts

A human papillomavirus (HPV) was isolated from the lesions of a patient (ML) bearing numerous hand common warts. This virus was compared with the well-characterized HPV found in typical plantar warts (plantar HPV). ML and plantar HPV DNAs have similar molecular weights (5.26 x 10(6) and 5.23 x 10(6), respectively) but were shown to be different by restriction enzyme analysis. When the cleavage products of both DNAs by endonuclease EcoRI, BamI, HpaI, or Hind were analyzed by electron microscopy, one, two, one, and four fragments were detected for ML HPV DNA instead of the two, one, two, and six fragments, respectively, detected for plantar HPV DNA. In contrast to plantar HPV DNA, a high proportion of ML HPV DNA molecules were resistant to these restriction enzymes. Most, if not all, of the molecules were either resistant to BamI and sensitive to EcoRI or sensitive to BamI and resistant to EcoRI. After denaturation and renaturation of the cleavage products of ML HPV DNA by a mixture of the two enzymes, the circular "heteroduplexes" formed showed one to three heterology loops corresponding to about 4 to 8% of the genome length. No sequence homology was detected between ML and plantar HPV DNAs by cRNA-DNA filter hybridization, by measuring the reassociation kinetics of an iodinated plantar HPV DNA in the presence of a 25-fold excess of ML HPV DNA, or by the heteroduplex technique. The two viruses had distinct electrophoretic polypeptide patterns and showed no antigenic cross-reaction by immunodiffusion or immunofluorescence techniques. Preliminary cRNA-DNA hybridization experiments, using viral DNAs from single or pooled plantar or hand warts, suggest that hand common warts are associated with viruses similar or related to ML HPV. The existence of at least two distinct types of HPVs that cause skin warts was demonstrated; they were provisionally called HPV type 1 and HPV type 2, with plantar HPV and ML HPV as prototypical viruses, respectively.     

Human papillomavirus type 29 (HPV-29), an HPV type cross-hybridizing with HPV-2 and with HPV-3-related types.

The cloning and partial characterization of human papillomavirus (HPV) type 29 is presented. By hybridization analyses, this virus appears to be related to HPV types associated with common warts and HPV types associated with flat warts.     

Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis.

Five human papillomavirus (HPV) DNAs from lesions of an epidermodysplasia verruciformis patient were cloned in lambda L 47: DNA of HPV 5, which predominated in the carcinoma; DNA of a variant type of HPV 8, which was not detected in the carcinoma DNA by Southern blot hybridization but only by cloning; and DNAs of three papillomaviruses that were isolated from warts. Southern blot and liquid phase DNA-DNA hybridization under stringent conditions showed that the three viruses from warts were new types, which we named HPVs 19, 20, and 25. These viruses cross-hybridized between 3 and 29% among themselves and with HPVs 5 and 8. After physical mapping with several restriction enzymes, the colinear genomes were aligned with HPV 8 DNA to define early and late regions. HPVs 8, 19, and 25 shared homology in different parts of their genomes.     

尖锐湿疣样本中HPV病毒的分子检测

调查男性和女性尖锐湿疣样本中人乳头瘤病毒（HPV）的检出率及病毒类型,为研发相关防治疫苗提供依据,以HPV 外壳蛋白DNA序列为模板设计特异引物,SSP－PCR扩增检测样本中HPV的感染率和病毒类型。收集了北京及邯郸市医院门诊尖锐湿疣样本22例,其中男性13例,女性9例。检测发现所有样本中存在着高浓度的HPV病毒DNA。男性样本中有5例感染HPV6型,6例感染HPV11型,2例为HPV6＋HPV11混合感染。女性样本中有3例感染HPV6型,2例感染HPV11型,4例为 HPV6＋HPV11混合感染。被诊断为宫颈湿疣的4位女性还在其含宫颈粘膜脱落细胞的样本中检出了HPV16、HPV18、HPV33、HPV35、HPV45、HPV54、HPV56或HPV58等高危险型病毒类型。所有检测到的HPV病毒DNA片段均TA克隆并将测定的DNA序列存入了国际基因数据库GenBank（DQ003066－DQ003079）。调查没有在单纯的男女尖锐湿疣组织块中检测到除HPV6和HPV11以外的其他HPV类型。该研究建立了灵敏可靠的HPV分子检测及分型方法,尖锐湿疣中HPV的检出率达100％。 本研究初步结果显示导致男女尖锐湿疣的HPV病毒类型没有显著差异,主要为HPV6及HPV11型。     

Characterization of human papillomavirus type 13 from focal epithelial hyperplasia Heck lesions.

Focal epithelial hyperplasia Heck lesions of a Turkish patient were shown to contain papillomavirus-specific DNA, which was molecularly cloned into bacteriophage lambda. It proved to be related to human papillomavirus (HPV) type 6 DNA and HPV type 11 DNA. Reassociation kinetics revealed a cross-hybridization of 4 and 3%, respectively. There was no cross-reactivity with HPV type 1, 2, 3, 4, 5, 8, or 10. This papillomavirus type will be referred to as HPV type 13. The DNA was characterized by cleavage with several restriction enzymes, and the cleavage sites were physically mapped. Papules from two additional cases of Morbus Heck contained HPV type 13 DNA as shown by Southern blot hybridization and by the characteristic cleavage patterns. This may indicate that HPV type 13 is more frequently associated with focal epithelial hyperplasia Heck than are other HPV types.     

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.     

Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively. These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12. The HPV-3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322. The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA. The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps. Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA. Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA. Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-1a, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-11a, respectively). One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular evidence for multiple Toxoplasma gondii infections in individual patients in England and Wales: public health implications

We sought to determine the SAG2 genotypes of Toxoplasma gondii associated with cases of acute human toxoplasmosis in England and Wales. The samples examined were collected from a wide range of cases including congenital infections, AIDS and immunosuppressed patients and were derived from a number of different tissues. Parasite DNA was detected by PCR amplification without the need for prior template purification, and SAG2 genotype was determined by both restriction enzyme analysis and direct DNA sequencing of the PCR amplification products. Parasites of both SAG2 type I and type II genotypes were seen with approximately equal frequency amongst the samples examined. Neither of these genotypes was found to be more frequently associated with a particular clinical presentation or sample tissue. Unexpectedly, we found clear evidence of mixed (SAG2 type I+type II) infections in approximately the same number of samples as were seen to be associated with either type I or II alone. Our use of direct DNA sequencing rather than simple restriction analysis was essential for the detection of mixed infections since incomplete restriction digestion of samples containing a single parasite type was occasionally observed. It is possible that the presence of more than one type of parasite in single samples might be related to our recent demonstration that mixtures of SAG2 type I and type II parasites are present in a significant proportion of commercial meat preparations. Moreover, the presence of mixed infections in single patients might offer a direct molecular method of assessing risk factors for infection.     

Identification of a Novel Human Papillomavirus,Type HPV199, Isolated from a Nasopharynx and Anal Canal,and Complete Genomic Characterization of Papillomavirus Species Gamma-12

The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.     

Functional analyses reveal an important role for tyrosine residues in the staphylococcal multidrug efflux protein QacA

Background

Human papillomavirus (HPV) tests are crucial diagnostic tools for the prevention of neoplastic lesions of the uterine cervix. However most commercial methods are designed to detect high-risk (HR) HPV types and a limited selection of low-risk ones, thus missing a fair number of intermediate/low-risk types. As a result, many HPV infections remain undiagnosed, generating distrust in virological diagnosis among gynaecologists, who continue to rely preferentially on cytological and colposcopic findings.

Results

In this study, we tested 6,335 consecutive clinical samples, most of them from Italian patients with cytological abnormalities. The samples, collected in 2000–2007, were analyzed using PCR amplification of a 173–206 bp (depending on HPV type) conserved region in the L1 open reading frame, restriction endonuclease analysis and, where required, sequence analysis for type determination. Analysis of a smaller male sample and long term follow-up of a few female subjects was also performed. A total of 2,161 samples tested positive for HPV DNA (32.1%); 21.3% of them were mixed infections. Overall, 59 known and 2 unknown HPV types were detected. Their relative prevalence was calculated; notably, types not clearly identifiable using the most common commercial method accounted for 36% of infections. Clinical findings associated with the underdiagnosed types ranged from H-SIL to low-grade abnormalities, although none of these infections resulted in invasive cancer.

Conclusion

Given the high prevalence of some underdiagnosed HPV types in the population (principally HPV53, HPV66, HPV84, and HPV87) and their frequent association with cytological abnormalities, techniques capable of detecting and typing them would prove extremely useful.     

Characterization of Two Novel Gammapapillomaviruses,HPV179 and HPV184, Isolated from Common Warts of a Renal-Transplant Recipient

Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient’s facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients’ facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens, suggesting its dual tissue tropism.     

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.     

Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity.

The DNA of human papillomavirus (HPV) obtained from a pool of plantar warts is cleaved by bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) restriction endonucleases at one and four specific sites, respectively. These sites were localized on the previously established cleavage map of HPV DNA, using the Hind, HindIII, HpaI, and EcoRI endonuclease restriction sites as reference. The four HpaII sites were mapped, clockwise, at 1.4, 41.1, 44.3, and 52.8% of the genome length from the unique BamI cleavage site taken as point zero. The HpaII site mapped at 1.4% of the genome length was absent in 40 to 50% of the molecules, thus showing a genetic heterogeneity of HPV DNA.     

Human papillomaviruses in anogenital warts in children: typing by in situ hybridisation.

OBJECTIVE--To identify the types of human papillomaviruses found in anogenital warts in children and to relate these to clinical and social information. DESIGN--In situ hybridisation using biotin labelled DNA probes to 11 types of human papillomavirus was performed on biopsy specimens from 17 children with anogenital warts. SETTING--Nuffield department of pathology and the department of dermatology, Oxford. PATIENTS--Children in one group were referred by general practitioners or paediatricians to the dermatology department, where biopsies were performed. The other children were seen in four different hospitals, and biopsy specimens were submitted to the laboratory at the physician''s or pathologist''s request. RESULTS--Of the 17 biopsy specimens, 10 contained cells positive with a probe to a genital human papillomavirus type (types 6 or 11), while six were positive with a skin virus type (types 2 or 3). One was negative. The virus type present bore no relation to the site or appearance of the warts. The virus type did, however, appear to correlate with groups of children. Skin types were commoner in older children (over 4 years), in those with a relative who had skin warts, and in children with warts elsewhere; there was no relation with the child''s sex and no suspicion of sexual abuse in these children. These circumstances suggested non-sexual transmission, such as autoinoculation. In contrast, genital types were commoner in girls, in children under 3 years, in children with relatives with genital warts, and in those with no warts elsewhere. Nevertheless, there was suspicion or evidence of sexual abuse in only half these children, suggesting that other routes of transmission--for example, perinatal--might have been implicated. CONCLUSION--Anogenital warts in children may contain either skin or genital wart virus type. Although the type of human papillomavirus present may give some indication of the likely mode of transmission, this can be interpreted only in conjunction with all available clinical and social information. The type of virus does not provide proof of the presence or absence of sexual transmission.     

HPV 6b L1基因原核表达系统的构建及鉴定

克隆、构建人乳头瘤病毒6b型(HPV6b)L1基因重组质粒,并进行表达和鉴定。用PCR方法,从尖锐湿疣标本中扩增出HPV6b型L1基因,构建重组克隆质粒pblue-HPV6bL1,测序分析其基因变异。将本地株HPV6b型L1基因酶切后连接到原核表达质粒pBAD,构建原核表达系统pBAD-HPV6bL1/Top10,酶切鉴定证明重组质粒的正确性。经L-Arobinose诱导后表达HPV6b型L1蛋白,利用SDS-PAGE对表达产物进行鉴定。经酶切及序列分析鉴定,重组质粒pBAD-HPV6bL1构建成功,诱导后能够表达L1融合蛋白。HPV6bL1重组质粒构建成功,并获得HPV6bL1蛋白,为该蛋白的功能及HPV的基因工程疫苗研究提供了物质基础。     

The interaction of high and low-risk human papillomavirus genotypes increases the risk of developing genital warts: A population-based cohort study

Cervical cancer is among the most common type of cancers in women and is associated with human papillomavirus (HPV) infection. Genital warts are also reported to be linked with HPV infection types 11 and 6. In turn, clinical characteristics and morphological features of warts may be useful in the prediction of prognosis and in making treatment decisions. Thus, we have investigated the association of high and low-risk HPVs genotype with genital wart risk, as well as pathological and cytological information in cases recruited from a population-based cohort study of 1380 patients. Patients infected with HPV genotype 6 or 11 had an increased risk of having warts, with OR of 2.34 (95% CI: 0.955-5.737, P = 0.06). Also, this association was enhanced in the presence of high plus low-risk HPV for having genital wart (OR: 2.814; 95%: 1.208-6.55, P = 0.017) and cases having high-risk HPV (OR: 2.329; 95% CI: 1.029-5.269, P = 0.042). Moreover, we observed patients with genital warts having CIN2/3, indicating the importance of informing the physician to the patient to prevent more severe lesions. Our data demonstrated that patients with both low/high-risk HPV types had an increased risk of developing genital warts and persistent infection with HPV was a necessary precursor for the increase in cervical lesions.     

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.     

Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis.

The DNAs of the human papillomaviruses (HPVs) associated with the benign lesions of two patients suffering from epidermodysplasia verruciformis (patients JD and JK) were analyzed by using 12 restriction endonucleases. None of the restriction endonucleases were one-cut enzymes for the HPV DNA obtained from patient JD, referred to as the prototypical HPV-5, whereas five of them were one-cut enzymes for the DNA of the major virus found in patient JK, referred to as HPV-9. The molecular cloning of the two fragments resulting from the cleavage of HPV-5 DNA by endonuclease HindIII and of the single fragment obtained after treatment of HPV-9 DNA with endonuclease BamHI was performed in Escherichia coli after the fragments were inserted in plasmid pBR322. A cleavage map of the two cloned genomes was constructed. Little sequence homology (4 to 5%) was detected between HPV-5 and HPV-9 DNAs by DNA-DNA hybridization experiments in liquid phase at saturation; this homology was reproducibly higher than that (2 to 3%) detected under the same conditions between these DNAs and HPV-1a DNA. In addition, blot hybridization experiments performed under stringent conditions showed no or little sequence homology between the DNAs of HPV-5 and HPV-9 and those of HPV prototypes of types 1, 2, 3, 4, and 7 associated with skin warts. These results confirm that HPV-5 and HPV-9 are two distinct HPV types.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Human papillomavirus genotyping by multiplex pyrosequencing in cervical cancer patients from India

Cervical cancer is a leading cause of cancer-related deaths among women in India.Human papillomavirus (HPV) infection is the causative agent of cervical cancer; and infection with the high-risk genotypes, predominantly HPV16 and 18,is the biggest risk factor.Vaccines targeting HPV16 and 18 have been found to confer protection in large- scale clinical trials.HPV genotyping has traditionally been carried out to screen the population "at risk" using indirect methods based on polymerase chain reaction (PCR) using consensus primers combined with various DNA hybridization techniques,and often followed by the sequencing of candidate products.Recently,a high-throughput and direct method based on DNA sequencing has been described for HPV genotyping using multiplex pyrosequencing. We present a pilot study on HPV genotyping of cervical cancer and non-malignant cervical samples using multiplex pyrosequencing.Using genomic DNA from cell lines,cervical biopsies,surgical tissues or formalin-fixed,paraffin- embedded tissue samples,we could successfully resolve 6 different HPV types out of the 7 tested,with their prevalence found to be in agreement with earlier reports. We also resolved coinfections with two different HPV types in several samples. An HPV16 genotype with a specific and recurrent sequence variation was observed in 8 cancer samples and one non-malignant sample. We find this technique eminently suited for high-throughput applications,which can be easily extended to large sample cohorts to determine a robust benchmark for HPV genotypes prevalent in India.