Related Articles

The genomes of two novel human papillomavirus (HPV) types, HPV68 and HPV70, were cloned from a low-grade cervical intraepithelial neoplasia and a vulvar papilloma, respectively, and partially sequenced. Both types are related to HPV39, a potentially oncogenic virus. HPV68 and HPV70 were also detected in genital intraepithelial neoplasia from three patients and one patient, respectively. Comparison with sequence data in the literature indicates that the subgenomic ME180-HPV DNA fragment, cloned from a carcinoma cell line, corresponds to an HPV68 subtype and that several HPV DNA fragments amplified by PCR from genital neoplasia represent worldwide distributed variants of HPV68 and HPV70.

Naturally occurring genetic variants of human papillomavirus type 16 (HPV16) are common and have previously been classified into 4 major lineages; European-Asian (EAS), including the sublineages European (EUR) and Asian (As), African 1 (AFR1), African 2 (AFR2), and North-American/Asian-American (NA/AA). We aimed to improve the classification of HPV16 variant lineages by using a large resource of HPV16-positive cervical samples collected from geographically diverse populations in studies on HPV and/or cervical cancer undertaken by the International Agency for Research on Cancer. In total, we sequenced the entire E6 genes and long control regions (LCRs) of 953 HPV16 isolates from 27 different countries worldwide. Phylogenetic analyses confirmed previously described variant lineages and subclassifications. We characterized two new sublineages within each of the lineages AFR1 and AFR2 that are robustly classified using E6 and/or the LCR. We could differentiate previously identified AA1, AA2, and NA sublineages, although they could not be distinguished by E6 alone, requiring the LCR for correct phylogenetic classification. We thus provide a classification system for HPV16 genomes based on 13 and 32 phylogenetically distinguishing positions in E6 and the LCR, respectively, that distinguish nine HPV16 variant sublineages (EUR, As, AFR1a, AFR1b, AFR2a, AFR2b, NA, AA1, and AA2). Ninety-seven percent of all 953 samples fitted this classification perfectly. Other positions were frequently polymorphic within one or more lineages but did not define phylogenetic subgroups. Such a standardized classification of HPV16 variants is important for future epidemiological and biological studies of the carcinogenic potential of HPV16 variant lineages.

Background

Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.

Methods

A reference set of 62 HPV16 complete genome sequences was established and used to examine patterns of evolutionary relatedness amongst variants using a pairwise identity heatmap and HPV16 phylogeny. A BLAST-based algorithm was developed to impute complete genome data from partial sequence information using the reference database. To interrogate the oncogenic risk of determined and imputed HPV16 SNPs, odds-ratios for each SNP were calculated in a case-control viral genome-wide association study (VWAS) using biopsy confirmed high-grade cervix neoplasia and self-limited HPV16 infections from Guanacaste, Costa Rica.

Results

HPV16 variants display evolutionarily stable lineages that contain conserved diagnostic SNPs. The imputation algorithm indicated that an average of 97.5±1.03% of SNPs could be accurately imputed. The VWAS revealed specific HPV16 viral SNPs associated with variant lineages and elevated odds ratios; however, individual causal SNPs could not be distinguished with certainty due to the nature of HPV evolution.

Conclusions

Conserved and lineage-specific SNPs can be imputed with a high degree of accuracy from limited viral polymorphic data due to the lack of recombination and the stochastic mechanism of variation accumulation in the HPV genome. However, to determine the role of novel variants or non-lineage-specific SNPs by VWAS will require direct sequence analysis. The investigation of patterns of genetic variation and the identification of diagnostic SNPs for lineages of HPV16 variants provides a valuable resource for future studies of HPV16 pathogenicity.

Persistent infection by specific oncogenic human papillomaviruses (HPVs) is established as the necessary cause of cervix cancer. DNA sequence differences between HPV genomes determine whether an HPV has the potential to cause cancer. Of the more than 100 HPV genotypes characterized at the genetic level, at least 15 are associated, to varying degrees, with cervical cancer. Classification based on nucleotide similarity places nearly all HPVs that infect the cervicovaginal area within the α-PV genus. Within this genus, phylogenetic trees inferred from the entire viral genome cluster all cancer-causing types together, suggesting the existence of a common ancestor for the oncogenic HPVs. However, in separate trees built from the early open reading frames (ORFs; i.e. E1, E2, E6, E7) or the late ORFs (i.e. L1, L2), the carcinogenic potential sorts with the early region of the genome, but not the late region. Thus, genetic differences within the early region specify the pathogenic potential of α-HPV infections. Since the HPV genomes are monophyletic and sites are highly correlated across the genome, diagnosis of oncogenic types and non-oncogenic types can be accomplished using any region across the genome. Here we review our current understanding of the evolutionary history of the oncogenic HPVs, in particular, we focus on the importance of viral genome heterogeneity and discuss the genetic basis for the oncogenic phenotype in some but not all α-PVs.

Among the more than one hundred formally described human papillomavirus (HPV) types, 18 are referred to as high-risk HPV types due to their association with anogenital cancer. Despite pathogenic similarities, these types form three remotely related taxonomic groups. One of these groups is called HPV species 9 and is formed by HPV-16, the most common and best-studied type, together with HPV-31, -33, -35, -52, -58, and -67. Previous worldwide comparisons of HPV-16 samples showed about 2% nucleotide diversity between isolates, which were subsequently termed variants. The distribution of divergent variants has been found to correlate frequently with the geographic origin and the ethnicity of the infected patients and led to the concept of unique African, European, Asian, and Native American HPV-16 variants. In the current study, we address the question of whether geography and ethnicity also correlate with sequence variations found for HPV-31, -35, -52, and -58. This was done by sequencing the long control region in samples derived from Europe, Asia, and Africa, and from immigrant populations in North and South America. We observed maximal divergence between any two variants within each of these four HPV types ranging from 1.8 to 3.6% based on nucleotide exchanges and, occasionally, on insertions and deletions. Similar to the case with HPV-16, these mutations are not random but indicate a relationship between the variants in form of phylogenetic trees. An interesting example is presented by a 16-bp insert in select variants of HPV-35, which appears to have given rise to additional variants by nucleotide exchanges within the insert. All trees showed distinct phylogenetic topologies, ranging from dichotomic branching in the case of HPV-31 to star phylogenies of the other three types. No clear similarities between these types or between these types and HPV-16 exist. While variant branches in some types were specific for Europe, Africa, or East Asia, none of the four trees reflected human evolution and spread to the extent illustrated by HPV-16. One possible explanation is that the rare HPV types that we studied spread and thereby diversified more slowly than the more abundant HPV-16 and may have established much of today's variant diversity already before the worldwide spread of humans 100,000 years ago. Most variants had prototypic amino acid sequences within the E6 oncoprotein and a segment of the L1 capsid protein. Some had one, two, or three amino acid substitutions in these regions, which might indicate biological and pathogenic diversity between the variants of each HPV type.

We examined the genomic diversity of human papillomavirus type 6 (HPV-6) and HPV-11 isolates from different parts of the world by comparing the nucleotide sequences of part of the long control region of three reference clones and 62 HPV-6 and 40 HPV-11 isolates from Africa, Europe, Asia, and North and South America. The genomic sequence of the HPV-6b reference type had to be amended by inclusion of a 94-bp segment, which is also present with minor differences in HPV-6a. Aside from two small inserts typical of all variants related to HPV-6a and three inserts found in HPV-11 variants, no major alterations to the size of the long control regions of these viruses were observed. This corrects the previous impression that these two HPV types are highly polymorphic. Altogether, 19 HPV-6 and 10 HPV-11 variant genomes could be distinguished, and most of the differences were due to point substitutions. The variants of either type were continuously connected in phylogenetic trees rather than clustered separately into subtype groups. Thirteen mutations, namely, the two HPV-6a inserts and 11 substitutions in HPV-6 or HPV-11 variants, reduced the dissimilarity between the types, but they bridged only a small fraction of the genetic distance between the two types. Genomes more obviously intermediate between HPV-6 and HPV-11 were not found and probably do not exist any more.(ABSTRACT TRUNCATED AT 250 WORDS)

Background

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.

Principal Findings

We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.

Conclusions

These data suggest that the alpha-HPV E6 proteins' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.

In this study, we have examined intratype human papillomavirus (HPV) sequence variation in a worldwide collection of cervical specimens. Twelve different HPV types including HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM9/PAP238A (recently designated HPV-73), and a novel partial genomic HPV sequence designated MM4/Wl3B were analyzed in this study. Cervical specimens were collected as part of epidemiological investigations conducted in New Mexico and an international study of invasive cervical cancer (IBSCC). Specimens from several countries including Argentina, Brazil, Bolivia, Benin, Cuba, Colombia, Chile, Germany, Mali, Panama, Paraguay, Spain, Algeria, Uganda, Guinea, Tanzania, Indonesia, Philippines, Thailand, and the United States were evaluated. Specimen DNAs were subjected to amplification with the MY09/11 L1 consensus PCR system. The PCR products were cloned, and an approximately 410-bp region in the L1 open reading frame was sequenced from 146 specimens (approximately 60,000 bp). Within a single HPV type, nucleotide diversity varied between 0.2 and 2.9% (i.e., between any pair of variants) and the majority of nucleotide changes were synonymous (amino acid conserving). These data provide information pertinent to HPV diagnostic probe development and are potentially relevant to future rational vaccine strategies. Similarly, amino acid diversity varied between 0 and 5.1%. Some of these amino acid changes may represent markers of intertype evolutionary relationships. Presuming that HPVs have evolved under the same constraints as their corresponding hosts, the limited genetic diversity observed for all HPVs studied to date may reflect an evolutionary bottleneck occurring in both virus and host populations.

Human papillomavirus type 18 (HPV18) and HPV45 account for approximately 20% of all cervix cancers. We show that HPV18, HPV45, and the recently discovered HPV97 comprise a clade sharing a most recent common ancestor within HPV α7 species. Variant lineages of these HPV types were classified by sequence analysis of the upstream regulatory region/E6 region among cervical samples from a population-based study in Costa Rica, and 27 representative genomes from each major variant lineage were sequenced. Nucleotide variation within HPV18 and HPV45 was 3.82% and 2.39%, respectively, and amino acid variation was 4.73% and 2.87%, respectively. Only 18 nucleotide variations, of which 10 were nonsynonymous, were identified among three HPV97 genomes. Full-genome comparisons revealed maximal diversity between HPV18 African and non-African variants (2.6% dissimilarity), whereas HPV18 Asian-American [E1 (AA)] and European (E2) variants were closely related (less than 0.5% dissimilarity); HPV45 genomes had a maximal difference of 1.6% nucleotides. Using a Bayesian Markov chain Monte Carlo (MCMC) method, the divergence times of HPV18, -45, and -97 from their most recent common ancestors indicated that HPV18 diverged approximately 7.7 million years (Myr) ago, whereas HPV45 and HPV97 split off around 5.7 Myr ago, in a period encompassing the divergence of the great ape species. Variants within the HPV18/45/97 lineages were estimated to have diverged from their common ancestors in the genus Homo within the last 1 Myr (<0.7 Myr). To investigate the molecular basis of HPV18, HPV45, and HPV97 evolution, regression models of codon substitution were used to identify lineages and amino acid sites under selective pressure. The E5 open reading frame (ORF) of HPV18 and the E4 ORFs of HPV18, HPV45, and HPV18/45/97 had nonsynonymous/synonymous substitution rate ratios (dN/dS) over 1 indicative of positive Darwinian selection. The L1 ORF of HPV18 genomes had an increased proportion of nonsynonymous substitutions (4.93%; average dN/dS ratio [M3] = 0.3356) compared to HPV45 (1.86%; M3 = 0.1268) and HPV16 (2.26%; M3 = 0.1330) L1 ORFs. In contrast, HPV18 and HPV16 genomes had similar amino acid substitution rates within the E1 ORF (2.89% and 3.24%, respectively), while HPV45 E1 was highly conserved (amino acid substitution rate was 0.77%). These data provide an evolutionary history of this medically important clade of HPVs and identify an unexpected divergence of the L1 gene of HPV18 that may have clinical implications for the long-term use of an L1-virus-like particle-based prophylactic vaccine.

Using low-stringency Southern blot analysis and cloning in lambda bacteriophage, two new human papillomavirus types (HPV-43 and HPV-44) were identified and their DNAs were cloned from vulvar tissues. The isolates were characterized by restriction endonuclease mapping and shown to be new HPV types on the basis of their minimal hybridization with all other known HPV types at high stringency. Both HPVs are most closely related to types 6, 11, and 13. HPV-43 did not exhibit any cross-reactivity with these HPV types at high stringency. HPV-44 showed minimal cross-reactivity to HPV-13, which was in the range of 20 to 25% according to liquid hybridization analysis. The deduced genomic organization of each of the two new HPVs was colinear with HPV-6b. Prevalence studies revealed that HPV-43 and HPV-44 together were found in 6 of 439 normal cervical tissues, in 8 of 195 cervical intraepithelial neoplasms, but in none of 56 cervical cancers tested thus far.

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Background

Infection with multiple human papillomavirus (HPV) types is common. However, it is unknown whether viral DNA load is related to the coexistence of other types.

Methods

Study subjects were 802 and 303 women who were positive for HPV16 and HPV18, respectively, at enrollment into the Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study. HPV16 and HPV18 E7 copies per nanogram of cellular DNA in cervical swab samples were measured by real-time polymerase chain reaction in triplicate.

Results

Concurrent coinfection was common in this population of women with minor cervical lesions; multiple HPV types were detected in 573 (71.4%) of 802 HPV16-positive women and 227 (74.9%) of 303 HPV18-positive women. The adjusted odds ratio associating coinfection with per 1 log unit increase in HPV16 DNA load was 0.78 (95% confidence interval (CI), 0.68-0.89); it was 0.64 (95% CI, 0.52-0.79) for a similar analysis of HPV18 DNA load. Women with, compared to without, coinfection of A9 species possessed a significantly lower HPV16 DNA load (p < 0.001) whereas women with, compared to without, coinfection of A7 species types possessed a significantly lower HPV18 DNA load (p = 0.001). A trend of decrease in HPV16 DNA load with increasing number of the coexisting non-HPV16 A9 species types was statistically significant (p for trend = 0.001).

Conclusion

Coinfection with other types was associated with lower HPV16 and HPV18 DNA load. The extent of reduction was correlated to phylogenetic distance of the coexisting types to HPV16 and HPV18, respectively.

Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1− mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5′ mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1− genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.

Aims—To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions.

Methods—One hundred and seventy six cytological specimens from women with different cervical lesions were investigated. For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Semiquantitative determinations were achieved in relation to an external reference titration curve.

Results—HPV DNA was detected in 60.2% of the samples. HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45. HPV-11 was not detected. HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease. Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease.

Conclusions—Only HPV-16 load appears to be associated with the severity of cervical disease.

Key Words: human papillomavirus distribution • viral load • cervical lesions

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.

The genome organization of the novel human papillomavirus type 108 (HPV108), isolated from a low-grade cervical lesion, deviates from those of other HPVs in lacking an E6 gene. The three related HPV types HPV103, HPV108, and HPV101 were isolated from cervicovaginal cells taken from normal genital mucosa (HPV103) and low-grade (HPV108) and high-grade cervical (HPV101) intraepithelial neoplasia (Z. Chen, M. Schiffman, R. Herrero, R. DeSalle, and R. D. Burk, Virology 360:447-453, 2007, and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. Expression of HPV108 E7 in organotypic keratinocyte cultures increases proliferation and apoptosis, focal nuclear polymorphism, and polychromasia. This is associated with irregular intra- and extracellular lipid accumulation and loss of the epithelial barrier. These alterations are linked to HPV108 E7 binding to pRb and inducing its decrease, an increase in PCNA expression, and BrdU incorporation, as well as increased p53 and p21CIP1 protein levels. A delay in keratin K10 expression, increased expression of keratins K14 and K16, and loss of the corneal proteins involucrin and loricrin have also been noted. These modifications are suggestive of infection by a high-risk papillomavirus.

Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-β signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-β expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-β and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.

Papillomaviruses are an ideal model system for the study of DNA virus evolution. On several levels, phylogenetic trees of papillomaviruses reflect the relationship of their hosts. Papillomaviruses isolated from remotely related vertebrates form major branches. One branch of human papillomaviruses (HPVs) includes an ape and two monkey papillomaviruses, possibly because the diversification of the viruses predated the separation of the infected-primate taxa. This hypothesis predicts that the root of the evolution of some if not all HPV types should point to Africa, since humans evolved from nonhuman primates in this continent. We tested this hypothesis and compared the genomic sequences of HPV type 18 (HPV-18) isolates from four continents. Diversity within HPV-18 correlates with patterns of the evolution and spread of Homo sapiens: HPV-18 variants, just like HPV-16 variants, are specific for the major human races, with maximal diversity in Africa. Outgroup rooting of the HPV-18 tree against HPV-45, which is closely related to HPV-18, identifies African HPV-18 variants at the root of the tree. The identification of an African HPV-45 isolate further reduces the evolutionary distance between HPV-18 and HPV-45. HPV-18 variants from Amazonian Indians are the closest relatives to those from Japanese and Chinese patients and suggest that a single point mutation in the phylogenetically evaluated genomic segment represents at least 12,000 years of evolution. We estimate that diversity within HPV-18 and probably within other HPV types evolved over a period of more than 200,000 years and that diversity between HPV types evolved over several million years.

Background

The variation of human papillomavirus (HPV) genes or HPV variants demonstrates different risks of cervical cancer. Mutation in the long control region (LCR) at YY1-motifs is one of the mechanisms for enhancing viral oncogene expression during the course of cancer cell progression. In Thai women, cervical cancers are almost always associated with HPV16 variant sub-lineage Asian (HPV16As); however, the mechanism involved remains elusive. The aim of this study was to understand further the oncogenic potential of HPV16As.

Methods

A total of 82 HPV16-positive specimens from Thai women were selected from formalin-fixed paraffin-embedded cervical tissues, and the full length E6 gene of each specimen was amplified and sequenced. LCRs of the HPV16As-positive cases were amplified and sequenced to analyze their polymorphisms. Transcriptional activities of the HPV16As LCRs were then compared with sub-lineage European (EUR), sub-lineage Asian-American 1 (AA1) and HPV16 prototype by insertion of the LCRs into the pGL3-Basic vector.

Results

The HPV16 DNA sequences were classified as HPV16 prototype (18.3%), Asian (As, 61%), Asian American-1 (AA1, 8.5%), European (EUR, 7.3%), Asian African-2 (AFR2, 3.7%) and Java-135C (J135C, 1.2%). The prevalence of HPV16As was 30% in low-grade squamous intraepithelial lesion (LSIL), while that in high-grade squamous intraepithelial lesion (HSIL) and squamous cell cervical carcinoma (SCC) were 63.9% and 66.7%, respectively, which demonstrates a significant association of HPV16As with the disease severity. LCR polymorphisms from 43 HPV16As positive cases were analyzed by PCR-sequencing. Thirty-eight nucleotide variation positions spanned nucleotide positions 7157–82. Ten new mutations found in the HPV16As LCRs were located predominantly at the enhancer and proximal to the 3’-end of the early promoter. The LCRs of the common HPV16As, EUR and AA1 showed 5, 13 and 23-fold higher activity than the HPV16 prototype LCR, while those of the new nucleotide variations of As showed 19 (As-sv1) and 30 (As-sv14) -fold higher activity than the HPV16 prototype.

Conclusions

HPV16As DNA sequence variation, especially at the proximal to early promoter in the LCR, enhances transcriptional activity. This could be one of the possible mechanisms for HPV16As-associated cervical cancer development.

Background

Human papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients.

Results

Whereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA.

Conclusions

The plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

HPV types differ profoundly in cervical carcinogenicity. For the most carcinogenic type, HPV16, variant lineages representing further evolutionary divergence also differ in cancer risk. Variants of the remaining 10-15 carcinogenic HPV types have not been well-studied.

In the first prospective, population-based study of HPV variants, we explored whether, on average, the oldest evolutionary branches within each carcinogenic type predicted different risks of ≥2-year viral persistence and/or precancer and cancer (CIN3+). We examined the natural history of HPV variants in the 7-year, 10,049-woman Guanacaste Cohort Study, using a nested case-control design. Infections were assigned to a variant lineage determined by phylogenetic parsimony methods based on URR/E6 sequences. We used the Fisher's combination test to evaluate significance of the risk associations, cumulating evidence across types.

Globally, for HPV types including HPV16, the p-value was 0.01 for persistence and 0.07 for CIN3+. Excluding HPV16, the p-values were 0.04 and 0.37, respectively. For HPV16, non-European viral variants were significantly more likely than European variants to cause persistence (OR = 2.6, p = 0.01) and CIN3+ (OR = 2.4, p = 0.004). HPV35 and HPV51 variant lineages also predicted CIN3+.

HPV variants generally differ in risk of persistence. For some HPV types, especially HPV16, variant lineages differ in risk of CIN3+. The findings indicate that continued evolution of HPV types has led to even finer genetic discrimination linked to HPV natural history and cervical cancer risk. Larger viral genomic studies are warranted, especially to identify the genetic basis for HPV16's unique carcinogenicity.

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

Persistent HPV infection plays a major role in cervical cancer. This study was undertaken to identify HPV types in a cohort of Indian women with locally advanced cervical cancer as well as to determine the physical state and/or site of viral integration in the host genome. Pretreatment biopsies (n = 270) from patients were screened for HPV infection by a high throughput HPV genotyping assay based on luminex xMAP technology as well as MY09/11 PCR and SPF1/2 PCR. Overall HPV positivity was observed to be 95%, with HPV16 being most common (63%) followed by infection with HPV18. Integration status of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay in a subset of samples positive for HPV16 and/or HPV18 (n = 86) and with an adequate follow-up. The data was correlated with clinical outcome of the patients. Integration of the viral genome was observed in 79% of the cases and a preference for integration into the chromosomal loci 1p, 3q, 6q, 11q, 13q and 20q was seen. Clinical data revealed that the physical state of the virus (integrated or episomal) could be an important prognostic marker for cervical cancer.

Cervical infections by approximately 15 cancer-associated (carcinogenic) human papillomavirus (HPV) genotypes cause virtually all cervical cancer and its immediate precursor lesions worldwide. Prophylactic vaccines against human papillomavirus (HPV) types HPV16 and HP18, which cause 70% of cervical cancer worldwide, hold great promise for reducing the burden of cervical cancer worldwide. However, current HPV vaccines prevent future infections and related cervical abnormalities and do not treat pre-existing HPV infections. In the U.S., HPV vaccine introduction should be considered in the context of a very successful cervical cancer screening program that has reduced the rates of cervical cancer by 75% or more. Thus, HPV vaccines will only prevent an incremental number of additional cervical cancers in the U.S. The introduction of HPV vaccines can also prevent other HPV-related sequelae, most importantly cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3), which precede the development of cervical cancer and require clinical follow-up and treatment. Examining data from 7 clinical centers in the U.S., the median age of CIN2/3 is typically between 25-30 years of age in 2007; if screen-detected CIN2/3 develops on average 5-10 years after the causal infection is acquired, HPV vaccination will only prevent a significant proportion of CIN2/3 if it is given to women before the age of 26 and more so if given to women 18 and younger. It is increasingly evident that prophylactic HPV vaccines will provide the greatest public health or population benefit only when delivered to adolescent women.

To assess the role of human papillomavirus virus (HPV) genetics in cervical lesions, we sequenced the E7 gene of HPV16, 31, or 73 from singly infected women who (1) cleared the infection quickly, (2) had type-specific persistent infection, or (3) progressed to CIN2 or worse lesions. Four of the 296 HPV16 E7 nucleotides were variable, compared with 7 of 296 for HPV31 E7 and 4 of 296 for HPV73 E7. While most of the polymorphisms in HPV31 and -73 resulted in non-synonymous amino acid changes, the polymorphisms in the HPV16 E7 resulted in synonymous changes. The lack of heterogeneity of HPV16 E7 suggests high evolutionary purifying selection that might be related to the unique carcinogenicity of HPV16.

Our aim was to study the phylogenetic relationships of all known papillomaviruses (PVs) and the possibility of establishing a supratype taxonomic classification based on this information. Of the many detectably homologous segments present in PV genomes, a 291-bp segment of the L1 gene is notable because it is flanked by the MY09 and MY11 consensus primers and contains highly conserved amino acid residues which simplify sequence alignment. We determined the MY09-MY11 sequences of human PV type 20 (HPV-20), HPV-21, HPV-22, HPV-23, HPV-24, HPV-36, HPV-37, HPV-38, HPV-48, HPV-50, HPV-60, HPV-70, HPV-72, HPV-73, ovine (sheep) PV, bovine PV type 3 (BPV-3), BPV-5, and BPV-6 and created a database which now encompasses HPV-1 to HPV-70, HPV-72, HPV-73, seven yet untyped HPV genomes, and 15 animal PV types. Three additional animal PVs were analyzed on the basis of other sequence data. We constructed phylogenies based on partial L1 and E6 gene sequences and distinguished five major clades that we call supergroups. One of them unites 54 genital PV types, which can be further divided into eleven groups. The second supergroup has 24 types and unites most PVs that are typically found in epidermodysplasia verruciformis patients but also includes several types typical of other cutaneous lesions, like HPV-4. The third supergroup unites the six known ungulate fibropapillomaviruses, the fourth includes the cutaneous ungulate PVs BPV-3, BPV-4, and BPV-6, and the fifth includes HPV-1, HPV-41, HPV-63, the canine oral PV, and the cottontail rabbit PV. The chaffinch PV and two rodent PVs, Micromys minutus PV and Mastomys natalensis PV, are left ungrouped because of the relative isolation of each of their lineages. Within most supergroups, groups formed on the basis of cladistic principles unite phenotypically similar PV types. We discuss the basis of our classification, the concept of the PV type, speciation, PV-host evolution, and estimates of their rates of evolution.