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We present an expansion of the classification of the family Papillomaviridae, which now contains 29 genera formed by 189 papillomavirus (PV) types isolated from humans (120 types), non-human mammals, birds and reptiles (64, 3 and 2 types, respectively). To accommodate the number of PV genera exceeding the Greek alphabet, the prefix "dyo" is used, continuing after the Omega-PVs with Dyodelta-PVs. The current set of human PVs is contained within five genera, whereas mammalian, avian and reptile PVs are contained within 20, 3 and 1 genera, respectively. We propose standardizations to the names of a number of animal PVs. As prerequisite for a coherent nomenclature of animal PVs, we propose founding a reference center for animal PVs. We discuss that based on emerging species concepts derived from genome sequences, PV types could be promoted to the taxonomic level of species, but we do not recommend implementing this change at the current time.

Approximately 35 years ago a role of human papillomaviruses (HPV) in cervical cancer has been postulated. Today it is well established that this very heterogeneous virus family harbours important human carcinogens, causing not only the vast majority of cervical, but also a substantial proportion of other anogenital and head and neck cancers. In addition, specific types have been linked to certain cutaneous cancers. In females, HPV infections on a global scale account for more than 50% of infection-linked cancers, in males for barely 5%. Vaccines against the high risk HPV types 16 and 18 represent the first preventive vaccines directly developed to protect against a major human cancer (cervical carcinoma). This review will cover some of the historical aspects of papillomavirus research; it tries briefly to analyze the present state of linking HPV to human cancers and will discuss some emerging developments.

Infection of a human T-cell leukaemia cell line (HSB-2) with HHV-6 led to the induction of exosome-like-particles attached to newly formed HHV-6 enveloped particles and to amplification of a 1642 bp molecule consisting of a partial human endogenous retrovirus (HERV)-E polymerase gene and repetitive sequences. We initiated an analysis of transcriptional patterns of predicted genes from HERV-E sequences in normal and malignant haematopoietic cells. Transcription patterns of regions corresponding to gag, pol and env genes at different chromosomal loci varied among cell types tested. Several specific transcripts were only observed in malignant haematopoietic cells and transcriptional activity varied among different malignant cell types. A transcript of 7.1 kb spanning the complete gag, pol and env gene region, originating from chromosome 8p23, was identified in normal peripheral blood cells and cells of the chronic myeloid leukaemia cell line K562. Our study describes new active HERV-E sequences and new loci throughout the human genome.

Rapid identification of human carcinogens before their dissemination into society, and exposure of worker and lay populations is an important goal of cancer research. Retroactively, verification of in-place human carcinogens is also required to target their removal, and other preventive and therapeutic strategies. The hierarchy of methods used historically for evaluation of carcinogenic potential is epidemiology > animal bioassays > mechanistic studies, and the focus has been on single agents that are genotoxic. However, mechanistic research has revealed several obligatory steps in carcinogenesis, tumor promotion, and progression that can now be used in screening studies with human cells in vitro and animal bioassays. These approaches should be combined with molecular epidemiology and molecular pathology to identify human carcinogens with more emphasis on evaluating combinations of suspect agents and mechanisms of action of epigenetic carcinogens.

One hundred eighteen papillomavirus (PV) types have been completely described, and a yet higher number of presumed new types have been detected by preliminary data such as subgenomic amplicons. The classification of this diverse group of viruses, which include important human pathogens, has been debated for three decades. This article describes the higher-order PV taxonomy following the general criteria established by the International Committee on the Taxonomy of Viruses (ICTV), reviews the literature of the lower order taxa, lists all known "PV types", and interprets their phylogenetic relationship. PVs are a taxonomic family of their own, Papillomaviridae, unrelated to the polyomaviruses. Higher-order phylogenetic assemblages of PV types, such as the "genital human PVs", are considered a genus, the latter group, for example, the genus "Alpha-Papillomavirus". Lower-order assemblages of PV types within each genus are treated as species because they are phylogenetically closely related, but while they have distinct genomic sequences, they have identical or very similar biological or pathological properties. The taxonomic status of PV types, subtypes, and variants remains unchanged and is based on the traditional criteria that the sequence of their L1 genes should be at least 10%, 2-10%, and maximally 2% dissimilar from one another.

Papillomaviruses cause certain common human cancers, most notably carcinoma of the cervix. The viral oncoproteins E6 and E7 are essential components in malignant conversion, although, in spite of being necessary, they are not sufficient for the development of the malignant phenotype. High risk HPV oncogenes fulfill dual functions in genome-harboring cells: their derived oncoproteins stimulate cell growth by pleiotropic effects. At the same time they act as progression factors by inducing mutations in host cell DNA and aneuploidy. The mechanism underlying the process towards malignant conversion, usually covering a long latency period between primary infection and cancer emergence, is presently not fully understood. It emerges, however, that cancer development depends on the interruption of at least two signalling cascades (labeled as CIF I and CIF II) that interfere with the function of viral oncoproteins (CIF I) and with the transcription of viral oncogenes (CIF II). Further modifications of host cell genes most likely mediate the escape from immune surveillance mechanisms of the host and the development of metastases., (Copyright 2000 Academic Press.)

The canine oral papillomavirus (COPV) is associated with oropharyngeal papillomatosis in dogs, coyotes, and wolves. We have determined the complete nucleotide sequence of COPV, the largest of all known PV genomes (8607 bp). The genomic architecture of the COPV genome is similar to that of other PVs except for a unique and large noncoding region of 1.5 kb between the end of the early region (E2) and the beginning of the late region (L2) and a small (345 bp) upstream regulatory region between the end of L1 and the beginning of E6. Although COPV displays a primarily mucosal tropism, the COPV nucleotide sequence showed the highest overall similarity to cutaneous papillomaviruses such as HPV-1, HPV-63, CRPV (cottontail rabbit PV), FdPV (Felis domesticus PV), and MnPV (Mastomys natalensis PV).

We have analyzed human papillomavirus (HPV) type 16 RNA expression in premalignant cervical lesions of different severity and in squamous cervical cancers by RNA-RNA in situ hybridization in order to find differences in the topographic distribution of viral RNA, which might correlate with the severity of disease. In the basal layer of low-grade squamous intraepithelial lesions (SIL) only weak transcription of viral early genes was observed. Signal intensity increased strongly in the more differentiated cells accompanied by high levels of HPV DNA replication. This pattern of viral gene expression, together with the onset of viral late transcription in the upper differentiated layer of the epithelium, most likely reflects the productive phase of viral infection. In contrast, in high-grade SIL viral transcription was comparatively strong in basal cells and evenly distributed throughout the undifferentiated epithelium. This difference of viral transcription in the basal layer of the respective lesions points to an altered regulation of viral gene expression which may be causally linked to the progression of precursor lesions. Evidence for disrupted expression of 3' early genes (E2, E4, and E5), analogous to the situation in HPV-DNA containing cervical carcinoma-derived cell lines, was not found in any of the HPV-16-positive premalignant lesions nor in the majority of cancers. The similarity of the viral transcription pattern of high-grade SIL and cancers suggests that additional host gene alterations are necessary for malignant progression.

To determine the prevalence of HPV 6, 11, 16, and 18 in a population without cytological or histological abnormalities, the cervical smears of women attending three clinics in Germany were screened over the past 5 years. The filter in situ hybridization method was used throughout. A total of 20,161 smears, taken from 11,667 women, were tested. When the results of only the first examination are considered, 8.8% (950/10,778) of women with normal cytology were positive for HPV DNA. If we divide the latter into age groups, 11% (852 HPV positive/7716) were below the age of 55 years and 3.2% (98 HPV positive/3062) were above this age. When the samples from patients who had undergone at least two examinations and remained cytologically negative during the 5-year period were examined (total, 2709 women), the HPV DNA positively increased to 34.7% (640/1862) for the sexually active age groups and to 9.0% (76/847) for those above 55 years of age. This study reveals that, although papillomaviral production is most pronounced in younger women, these infections are quite common in all age groups. During the period of investigation, 19 (0.65%) patients, who were diagnosed as cytologically negative at the first examination, progressed to carcinoma in situ or invasive carcinoma. Of these, 63.2% revealed a detectable HPV infection during the study period. The progression of HPV-positive women from normal cytology to CIN or cancer occurred at an annual frequency of 0.082%. With an infected lifespan of 45 years assumed, this results in a lifetime risk of 3.7%.

Substantial experimental evidence accumulated over the past 8 years has indicated an etiological role for specific human papillomavirus (HPV) types in anogenital cancer and its premalignant precursors. Virus infection and viral gene expression emerge as necessary but obviously not sufficient factors for cancer induction. Additional modifications of host cell genes appear to be required for malignant progression of infected cells. The expression of viral oncoproteins in cells infected by "high-risk" types (e.g., HPV 16, HPV 18), in contrast to "low-risk" types (e.g., HPV 6, HPV 11), results in chromosomal instability and apparently in accumulation of mutational events. These "endogenous" modifications seem to be most important in the pathogenesis of premalignant lesions and tumor progression. Exogenous mutagens should act as additional cofactors.

Herpes simplex virus (HSV) induces DNA amplification within the host cell genome, which is mediated by a set of six of seven HSV replication genes. The origin-binding protein (UL9) is dispensible. By the use of HSV mutants for the UL9 gene we show here that HSV can induce DNA amplification in the absence of lytic viral growth in contrast to replication-negative mutants for either the UL8 or UL52 gene used as control. The amplification-inducing potential of HSV may be relevant for the pathogenicity of the virus.

Infection with herpes simplex viruses (HSV) lead to a significant increase of the simian virus 40 (SV40) DNA content in the SV40-transformed hamster cell lines CO631 and Elona. Analysis of this gene-amplifying activity revealed (i) that it cosedimented with infectious herpesvirions in sucrose density gradients, (ii) that it was abolished by anti-HSV antibodies or (iii) by antiviral drugs acting on the HSV-induced DNA polymerase; and analysis of temperature-sensitive mutants showed that this DNA polymerase was an essential component of HSV-induced, gene-amplifying activity in SV40-transformed hamster cells.

An apparently helper-dependent parvovirus was isolated from a penile flat condylomatous lesion by inoculating the material into a culture of human fibroblasts, and by coinfecting these cells with adenovirus type 12. Upon addition of an adenovirus helper, the virus could be readily propagated in human KB cells. Analysis of its DNA revealed that it shares structural similarities with the DNA of the four defective parvoviruses (adeno-associated viruses, AAV) described so far, but that the cleavage pattern obtained after digesting the DNA with restriction enzymes is different from that of AAV 1-AAV 4. Nucleic acid hybridization data further support the assumption that the isolated defective parvovirus is not identical with the other four serotypes. When conditions of high stringency were used, only weak cross-reactivity was seen with the DNAs of AAV 1-AAV 4, whereas the latter showed strong cross-hybridization with each other. The organization of the DNA of the newly isolated parvovirus was investigated by mapping the cleavage sites of several restriction enzymes. It is proposed to designate the new isolate as AAV 5.

Time kinetics of AAV-5 DNA amplification induced by MNNG in cells of a Chinese hamster line (CO631) and a Syrian hamster line (Elona) were compared to kinetics of SV40 DNA amplification in these cells. AAV-5 DNA amplification starts in both lines about 8 hr following treatment of AAV-infected cells with MNNG. SV40 DNA amplification is induced only in CO631 cells. The time it becomes detectable varies but in no case was before 23 hr after MNNG treatment. In CO631 cells a second round of AAV DNA amplification takes place. It starts at times paralleling the MNNG-induced synthesis of SV40 DNA but appears to be independent of genotoxic treatment. Elona cells amplify neither SV40 DNA after exposure to chemical carcinogens nor AAV-5 DNA in untreated cells. In both lines combined treatment with MNNG and AAV-5 resulted in marked cytopathogenic changes and cell death. In the test systems used here viral DNA amplification did not lead to synthesis of infectious virus, thus, the viral cycle remained abortive.

SV40-transformed Chinese hamster cells (CO631) were treated with 7, 12-dimethylbenz[alpha]anthracene, 4-nitroquinoline-N-oxide, or benzo[alpha]pyrene, respectively, at sublethal concentrations. Infection of these cells with the helper-dependent parvovirus, AAV-5 immediately prior to, or at the time of exposure to the chemical carcinogens resulted in effective killing of cells exposed to the combination virus plus carcinogen, AAV infection without additional treatment did not have a significant effect on cell survival. AAV infections have recently been shown to efficiently inhibit gene amplification induced by various initiators and herpes simplex virus infections. Data may suggest that gene amplification is an adaptive cellular response to initiating events resulting in enhanced cell survival.

The protein compositions of full and empty particles of human papilloma viruses 1, 2, and 3 were compared by SDS-gel electrophoresis. The protein patterns revealed a considerable variation in the relative concentrations of three major proteins (VP2, 3, and 4) in individual preparations. In some cases, "heavy" and "light" full particles prepared from the same wart showed a similar variability in the concentrations of VP2, 3, and 4. The different protein patterns were interpreted as resulting from conversion of VP2 into VP3 and 4. The molecular relationship of these three proteins was confirmed by BrCN cleavage which led to corresponding oligopeptides. Electron micrographs of empty particles revealed that the only detectable protein components VP3 and 4 are present in typical capsomeres.

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens ("initiators") as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

A new type of defective parvovirus, tentatively designated as adeno-associated virus type 5 (AAV-5), is characterized as far as its proteins, its helper dependence, and its seroepidemiology are concerned. The protein analysis of AAV-5 in polyacrylamide gels demonstrated the presence of three structural polypeptides, corresponding to VP 1, VP 2, and VP 3 of other AAV types. The preparation of monoclonal antibodies against AAV-5 permitted the analysis of viral structural antigen expression by using adenovirus type 12 (Ad 12) or several herpes group viruses as helper viruses, respectively. AAV-5-infected cell cultures coinfected with either Ad 12, Herpes simplex virus (HSV), Cytomegalovirus (CMV), or Varicella Zoster virus (VZV) efficiently synthesize AAV-5 specific antigens. Epstein-Barr virus (EBV) and Herpesvirus saimiri, in contrast, provide only a very weak helper activity for AAV 5 antigen expression. The development of a specific ELISA test permitted screening of human sera for antibodies to AAV-5. Forty-five percent of 926 sera from all age groups and approximately 60% of the adult population reveal antibodies to structural components of this virus. The seroepidemiology differs from that reported for other AAV serotypes. Highest average titers against AAV-5 are observed in the age group between 15 and 20 years. Sera from patients with cervical carcinoma revealed average titers of antibodies well below those of age-matched control groups. Attempts to find higher antibody levels against AAV-5 in specific human diseases failed thus far.

Coinfection with HSV-1 and any of the three defective parvoviruses AAV-2, AAV-3, or AAV-5 reduces the HSV-1-induced amplification of SV40 DNA in the SV40-transformed hamster cell lines CO631 and Elona. Moreover, all three viruses significantly decrease HSV-1 replication. The effects are measurable to the same extent in both cell lines and are dependent on the quantity of AAV DNA molecules rather than on infectious AAV particles. It seems unlikely that the inhibitions are due to simple competition but result from complex interactions that involve the terminal sequences of AAV DNA.

Lymphotropic papovavirus (LPV) is a new member of the polyomavirus genus. Its host range in vitro is restricted to transformed cells of B-lymphocyte origin. Here the complete 5270-bp DNA sequence of LPV is presented. The LPV early region can encode a large T and a small t antigen but no middle T antigen and the late region can encode the three structural proteins VP1, VP2, and VP3. Based on sequence conservation of shared proteins LPV is equally related to both mouse polyomavirus (Py) and simian virus 40 (SV40) and represents a new distinct species of the polyomavirus genus. Sequence comparisons of LPV, SV40, and Py point out essential conserved sequence features of the polyomavirus genus more clearly than the comparison of only SV40 and Py. The most conserved proteins are VP1 with 42% and large T antigen with 28% of the amino acids conserved among the three viruses. Although least conserved the noncoding DNA sequences of LPV show significant homologies both to SV40 and Py (origin of viral DNA replication and putative early promoter). A 63-bp tandem repeat at the late side of the replication origin possibly represents a LPV enhancer element.

The complete nucleotide sequence of human papilloma virus type 11 (HPV11) DNA (7931 bp) was determined. HPV11 DNA which has been isolated from laryngeal papillomas and from genital warts (condylomata acuminata) shows a high degree of sequence homology to HPV6b (82%). The arrangement of open reading frames is very similar to HPV6b, the homology of the deduced amino acid sequences varies between 58 and 92%. Characteristic features of the noncoding region between the L1 and E6 open reading frames is an AT-rich domain of about 200 bp with extended stretches of alternating thymine-purine bases and a 12-bp inverted repeat element ACCG NNNN CGGT arranged in tandem upstream of the putative early promoter TATA box.

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.

Two new papillomaviruses, HPV 40 and HPV 57, were isolated from a PIN lesion and an inverted papilloma of the maxillary sinus, respectively. HPV 40 showed a 13% homology to HPV 7 by reassociation kinetics and HPV 57 showed a 17% homology to HPV 2 and 25% homology to HPV 27. Hybridization of the DNA of these papillomaviruses to a wide variety of different tumor biopsies revealed that HPV 40 was present in a few genital condylomata acuminata as well as in bowenoid lesions. HPV 57 DNA was present in an oral wart, a genital condyloma acuminatum, and verrucae vulgares lesions from two immunosuppressed patients.