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Human papillomavirus types 2 (HPV-2), HPV-27, and HPV-57, are three closely related viruses within the phylogenetic supergroup formed by the remotely related genital papillomaviruses. In contrast to this phylogenetic association, these three viruses are most often found in common warts at nongenital sites, but also occasionally in genital warts and mucosal lesions of the nasopharyngeal cavity. We studied the genomic diversity of HPV sequences in skin warts presumably caused by these viruses. These biopsies were sampled from 75 patients living in Germany, Japan, or Singapore. Among 27 warts with HPV-2, we found seven new genomic variants and among 32 with HPV-57, eight new variants. In both cases, we did not detect the original prototype genomes. In contrast, 13 of 16 warts with HPV-27 contained the prototype genome, and only one new variant was found in three patients. We did not find variants clearly intermediate between any two types, although HPV-2 and HPV-27 are among the most closely related of the extent HPV types. We also did not detect novel HPV types, although the samples were examined with polymerase chain reaction protocols that would have detected remotely related HPVs. So we propose that the phylogenetic group formed by HPV-2, HPV-27, and HPV-57 has no or only very are additional members. One of the HPV-57 variants found, HPV-57-G44, was most likely identical to the subtype HPV-57b, previously proposed to be associated with nasal neoplasia, but found here frequently in common skin warts. Our publication establishes a foundation for pathological and phylogenetic comparisons of HPV types in skin warts.

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.

Portions of the genome from two different papillomaviruses (PVs) of the Abyssinian Colobus monkey were sequenced and phylogenetically analyzed. This revealed that the major evolutionary separation between genital PVs and epidermodysplasia verruciformis-associated PVs (EV-PVs) hitherto found only in human papillomaviruses (HPVs) also exists in animal PVs. The sequence of the long control region (LCR) of Colobus monkey PV type 2 (CgPV-2) reveals a small size and an arrangement of potential cis-responsive elements typical of the EV-HPVs; namely four binding sites for the viral E2 protein, with one of them being located within the L1 gene, a cluster of nuclear factor I (NFI)- and AP-1-binding sites and a 50-bp sequence upstream of the E6 gene consisting only of the nucleotides A and T. This level of conservation of functional elements within the highly variable LCR suggests that CgPV-2 could be adopted as a model for studying human skin cancer associated with EV-HPVs. Although isolated from the same monkey species, the other Colobus monkey PV, CgPV-1, is a typical genital PV as shown by E1 and L1 sequence comparisons. The presence of these two major phylogenetic divisions of PVs in both human and monkey hosts strongly suggests that this diversification predated the evolutionary split between monkeys and apes. In other words, at least two different groups of PVs have been evolving separately in their respective primate hosts for more than 22 million years with only moderate sequence changes since their genesis.

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. A single HPV-11 variant was found in Africa, but otherwise, no significant correlations of HPV-6 or HPV-11 variants with geography or ethnicity of the patient cohort were found. Functional analysis of diverse enhancer variants showed activities that differed two-to threefold, and it must be considered that transcriptional differences may alter the biology or pathology of these viruses. Similar variants were found in lesions from anatomically different sites and in both benign and malignant lesions.

India has one of the world's highest incidences of oral cancer. The habit of chewing betel quid is widespread and is suspected to play a role in the etiology of this disease. Studies in many other countries have also pointed to a role for human papilloma-viruses (HPVs) in the etiology of some oral cancers. In this study we analyzed biopsies from 91 Indian oral cancer patients, most of whom were betel quid chewers, by PCR amplification and direct DNA sequencing. HPV DNA was detected in 74% of these lesions, of which 41% had multiple HPV infections. Among the lesions from different oral sites, lesions of the tongue had the highest rate (9 of 11) of HPV infection. These HPV prevalences are among the highest ever reported in oral cancers. As to individual HPV types, prevalences of HPV-6, HPV-11, HPV-16 and HPV-18 were 13%, 20%, 42% and 47%, respectively. No additional known or novel HPV types were detected. To understand the unexpectedly high prevalences of the "low-risk" types HPV-6 and HPV-11, we compared the subtypes and variants that were found in oral cancers against those from benign genital warts from the same patient population but found no differences. The high prevalence of HPV in the oral cancers of these Indian patients suggests that viral infection is an important etiological component, with betel quid probably causing additional mutagenic steps in the carcinogenic process.

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.

The identification and taxonomy of papillomaviruses has become increasingly complex, as approximately 70 human papillomavirus (HPV) types have been described and novel HPV genomes continue to be identified. Methods and corresponding DNA sequence data bases were designed for the reliable identification of mucosal HPV genomes from clinical specimens. HPVs are identified by the amplification of a fragment of the L1 region by consensus primer polymerase chain reaction (PCR) and subsequent hybridization or restriction fragment length polymorphism analysis. L1 PCR fragments may be further characterized by nucleotide sequencing. Conservation of 30 (of 151) predicted amino acids identifies HPV genomic fragments, and nucleotide sequence alignments allow calculation of their phylogenetic relatedness. Sequence differences > 10% from any known HPV type suggest a novel HPV type. Phylogenetic relationships with known HPV types may permit predictions of biology. With these criteria, 10 PCR fragments were identified that would qualify as new genital HPV types after complete genomic isolation.

Human papillomavirus type 27 (HPV-27) and HPV-2c are isolates of the same virus as judged by genomic sequence identity. In a 400-bp stretch of the long control region and the E6 and L1 genes no changes were found between HPV-27 and HPV-2c whereas 55 changes were found between HPV-27 and HPV-2a. This justifies the elevation of HPV-2c to type status and strongly suggests its identity with HPV-27. This reassessment makes HPV-27 one of the most abundant viruses associated with common warts.

Mastomys natalensis is a rodent of African origin afflicted with a very high incidence of skin tumors (keratoacanthomas and squamous carcinomas), which are associated with a papillomavirus, M. natalensis papillomavirus (MnPV). We have determined the genomic sequence of MnPV, which has a size of 7687 bp. The genomic organization is similar to that of other papillomaviruses, with open reading frames E6, E7, E1, E2, and E4 in the early and L2 and L1 in the late region. Due to an unusually large hinge region, the transcriptional activator E2 has a size of 542 amino acids rather than 400 to 460 amino acids, as in other papillomaviruses. An open reading frame E5 coding for a small hydrophobic membrane protein is missing, as is the case for some cutaneous human papillomaviruses (HPV). This fact, together with the composition of cis-responsive elements in its long control region and phylogenetic evaluation of segments of its E6, E1, and L1 genes, indicates a relationship of MnPV to the cottontail rabbit papillomavirus and several HPV types found in lesions of cutaneous epithelia, in particular to those that are associated with epidermodysplasia verruciformis. MnPV may be a useful model system for tumorigenesis of cutaneous epithelia in humans.

We have investigated the diversity of a hypervariable segment of the human papillomavirus type 16 (HPV-16) genome among 301 virus isolates that were collected from 25 different ethnic groups and geographic locations. Altogether, we distinguished 48 different variants that had diversified from one another along five phylogenetic branches. Variants from two of these branches were nearly completely confined to Africa. Variants from a third branch were the only variants identified in Europeans but occurred at lower frequency in all other ethnic groups. A fourth branch was specific for Japanese and Chinese isolates. A small fraction of all isolates from Asia and from indigenous as well as immigrant populations in the Americas formed a fifth branch. Important patterns of HPV-16 phylogeny suggested coevolution of the virus with people of the three major human races, namely, Africans, Caucasians, and East Asians. But several minor patterns are indicative of smaller bottlenecks of viral evolution and spread, which may correlate with the migration of ethnic groups in prehistoric times. The colonization of the Americas by Europeans and Africans is reflected in the composition of their HPV-16 variants. We discuss arguments that today's HPV-16 genomes represent a degree of diversity that evolved over a large time span, probably exceeding 200,000 years, from a precursor genome that may have originated in Africa. The identification of molecular variants is a powerful epidemiological and phylogenetic tool for revealing the ancient spread of papillomaviruses, whose trace through the world has not yet been completely lost.

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.

Human papillomavirus type 16 (HPV-16) is causally involved in the pathogenesis of genital neoplasia, but important details of the natural history of infection and disease are not yet understood. Many individual HPV-16 DNA isolates differ by characteristic point mutations. In a study of the HPV-16 variants from genital lesions of 32 married couples, HPV-16 was detected in both the husband and the wife in 8 couples. Of these, 4 demonstrated identical HPV-16 variants between husband and wife, and 4 had mismatching HPV-16 variants. Five of 31 biopsies showed simultaneous presence of two different HPV-16 variants. The data suggest that sexual transmission of HPV-16 does occur, but with low infectivity, and that HPV-16-related premalignant lesions are frequently polyclonal.

Papillomaviruses are attractive models for studying the molecular evolution of DNA viruses because of the large number of isolates that exhibit genomic diversity and host species and tissue specificity. To examine their relationship, we selected two amino acid sequences, one of 52 residues within the early gene E1 and the other of 44 residues within the late gene L1, which allowed insertion- and deletion-free alignment of all accessible papillomavirus sequences. We constructed phylogenetic trees from the amino acid and corresponding nucleotide sequences from 28 published and 20 newly determined animal and human papillomavirus (HPV) genomic sequences by using distance matrix, maximum-likelihood, and parsimony methods. The trees agreed in all important topological aspects. One major branch with two clearly separated clusters contained 11 HPV types associated with epidermodysplasia verruciformis. A second major branch had all the papillomaviruses involved in genital neoplasia and, in distant relationship, the cutaneous papillomaviruses HPV type 2a (HPV-2a), HPV-3, and HPV-10 as well as the "butcher's" papillomavirus HPV-7 and two simian papillomaviruses. Four artiodactyl (even-toed hoofed mammal) papillomaviruses, the cottontail rabbit papillomavirus, and avian (chaffinch) papillomavirus type 1 formed a third major branch. Last, four papillomaviruses exhibited little affinity to any of these three branches; these were the cutaneous types HPV-1a, HPV-4, and HPV-41 and B-group bovine papillomavirus type 4. The phylogeny suggests that some branches of papillomavirus evolution are restricted to particular target tissues and that a general process of long-term papillomavirus-host coevolution has occurred. This latter hypothesis is still conjectural because of bias in the current data base for human types and the paucity of animal papillomavirus sequences. The comparison of evolutionary distances for the most closely related types with those of 28 subtypes and variants of HPV-2, HPV-5, HPV-6, HPV-16, and HPV-18 supports the type as a natural taxonomic unit, with subtypes and variants being expressions of minor intratype genomic diversity similar to that found in the natural populations of all biological species. An exception to this seems to be HPV-2c, which has an evolutionary distance from HPV-2a of the intertype magnitude and may eventually have to be regarded as a distinct type. We describe an experimental approach that estimates the taxonomic and phylogenetic positions of newly identified papillomaviruses without viral isolation and complete genomic sequencing.(ABSTRACT TRUNCATED AT 400 WORDS)

We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographic location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their likely introduction into the New World suggest a very slow rate of viral evolution. The phylogenetic tree therefore probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races.

Genomic variability between different viral isolates provides a powerful epidemiological tool for verifying ultrasensitive diagnostic procedures, understanding infectious pathways in individuals and human populations, and studying viral evolution. The potential of this approach has not yet been exploited for the diagnosis of human papillomaviruses (HPVs) like HPV type 16 (HPV-16), which are involved in genital cancer. Toward this end, we amplified by polymerase chain reaction, cloned, and sequenced a 364-bp noncoding segment of the HPV-16 genome from cell lines, cervical biopsy specimens, and cervical smears. The HPV-16 genomes in the cell lines SiHa and CaSki showed an identical point mutation, and in the SiHa cell line it had an additional 38-bp deletion. Only 4 of 22 cervical lesions biopsied from patients at several hospitals in Singapore contained HPV-16 DNA with the prototype sequence, while the DNAs of the other 18 cervical lesions differed by 1 to 10 mutations. This excludes contaminations with cloned HPV-16 DNA as the source of this DNA. To test whether this diversity was a geographic idiosyncrasy, we analyzed 25 cervical biopsy specimens from Brazil. Eight of these contained the prototype sequence, while 17 were mutated. Altogether, 11 genomic variants were found in the Singaporean samples and 12 genomic variants were found in the Brazilian samples, and only 5 of these occurred identically in both cohorts. All variants could be connected to form a phylogenetic tree, with some branches being specific for each cohort. This suggests that the variants did not originate over a short period in the individual patient but, rather, evolved consecutively while spreading throughout humankind.(ABSTRACT TRUNCATED AT 250 WORDS)