Evolutionary dynamics of ten novel Gamma-PVs: insights from phylogenetic incongruence, recombination and phylodynamic analyses.

BACKGROUND: Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. All papillomaviruses have double stranded DNA genomes that are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. Here we use the sequences of ten novel Gammapapillomaviruses (Gamma-PVs) characterized in previous studies and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. RESULTS: We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago, before splitting into the five main lineages. The LCA of the Gamma-PVs at this time was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. CONCLUSION: Consequently, we report here phylogenetic tree incongruence without strong evidence of recombination.

Discovery, characterisation and genomic variation of six novel Gammapapillomavirus types from penile swabs in South Africa.

Six novel human papillomaviruses from penile swabs were characterised. Multiple full genome clones for each novel type were generated, and complete genome sizes were: HPV211 (7253bp), HPV212 (7208bp), HPV213 (7096bp), HPV214 (7357), HPV215 (7186bp) and HPV216 (7233bp). Phylogenetically the novel papillomaviruses all clustered with Gammapapillomaviruses: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. The novel HPV types demonstrated the classical genomic organisation of Gammapapillomavirusess, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gammapapillomavirusess the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. HPV212 had nine unique variants, HPV213 had five and HPV215 had four variants. Conserved domains observed among the novel types are the Zinc finger Binding Domain and PDZ domains. A retinoblastoma binding domain (pRB) binding domain in E7 protein was additionally identified in HPV214. This study expands the knowledge of the rapidly growing Gammapapillomavirus genus.

High prevalence of Gammapapillomaviruses (Gamma-PVs) in pre-malignant cutaneous lesions of immunocompetent individuals using a new broad-spectrum primer system, and identification of HPV210, a novel Gamma-PV type.

Genus Gammapapillomavirus (Gamma-PV) is the most diverse and largest clade within the Papillomaviridae family. A novel set of degenerate primers targeting the E1 gene was designed and further used in combination with the well-known CUT PCR assay to assess HPV prevalence and genus distribution in a variety of cutaneous samples from 448 immunocompetent individuals. General HPV, Gamma-PV and mixed infections prevalence were significantly higher in actinic keratosis with respect to benign and malignant neoplasms, respectively (p = 0.0047, p = 0.0172, p = 0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (p = 0.002). The full-length genome sequence of a novel putative Gamma-PV type was amplified by 'hanging droplet' long-range PCR and cloned. The novel virus, designated HPV210, clustered within species Gamma-12. This study provides an additional tool enabling detection of HPV infections in skin and adds new insights about possible early roles of Gamma-PVs in the development of cutaneous malignant lesions.

Prevalence and Epidemiologic Profile of Oral Infection with Alpha, Beta, and Gamma Papillomaviruses in an Asian Chinese Population.

Background: Knowledge of the prevalence of and risk factors for oral human papillomavirus (HPV) infection, especially cutaneous types, is limited. Methods: A population-based study using next-generation sequencing consecutively recruited asymptomatic individuals aged 18-64 years from a proportional sampling of the general population of Hong Kong, according to age groups, gender, and regions of residence. We examined associations of alpha-, beta-, and gamma-HPVs from oral rinse samples with participants' sociodemographics by logistic regression models. Results: The prevalence of oral HPV infection among 1426 ethnic Chinese was 15.5% (95% confidence interval [CI], 13.7%-17.5%), 2.5% (95% CI, 1.8%-3.5%), 11.9% (95% CI, 10.3%-13.6%), and 2.9% (95% CI, 2.1%-3.9%) for any type, alpha-, beta-, and gamma-HPV, respectively. Prevalence of any high-risk HPV was 0.8% (95% CI, 0.4%-1.4%), and that of HPV-16 was 0.4% (95% CI, 0.2%-0.8%). HPV-8 and HPV-98 were the most common beta types detected, while HPV-4 and HPV-SD2R were the most common gamma types. Prevalence of alpha- and beta/gamma-HPV infection showed a similar pattern of increase with age, and was higher in men than women. Smoking, drinking, oral sex, and more sexual partners were associated with alpha-HPV. Teeth brushing before sleep was protective for beta/gamma-HPVs. Discussion: The epidemiologic factors associated with oral infection with alpha-HPVs are different from those of beta/gamma-HPVs, suggesting different modes of acquisition and persistence.

Identification and characterization of two novel Gammapapillomavirus genomes in skin of an immunosuppressed Epidermodysplasia Verruciformis patient.

Two novel human gamma-papillomavirus genomes (HPV_MTS3, and HPV_MTS4) were isolated from the skin of an immunosuppressed, late-onset Epidermodysplasia Verruciformis patient and fully cloned. The L1 open reading frames of HPV_MTS3 and HPV_MTS4 were 77% and 91% identical to their closest HPV full genome isolates w18c39 and EV03c60, which belong to the species gamma-22and gamma-7 of the genus Gammapapillomavirus, respectively.

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Proteomic analysis of the gamma human papillomavirus type 197 E6 and E7 associated cellular proteins.

Gamma HPV197 was the most frequently identified HPV when human skin cancer specimens were analyzed by deep sequencing (Arroyo Muhr et al., Int. J. Cancer 136: 2546-55, 2015). To gain insight into the biological activities of HPV197, we investigated the cellular interactomes of HPV197 E6 and E7. HPV197 E6 protein interacts with a broad spectrum of cellular LXXLL domain proteins, including UBE3A and MAML1. HPV197 E6 also binds and inhibits the TP53 tumor suppressor and interacts with the CCR4-NOT ubiquitin ligase and deadenylation complex. Despite lacking a canonical retinoblastoma (RB1) tumor suppressor binding site, HPV197 E7 binds RB1 and activates E2F transcription. Hence, HPV197 E6 and E7 proteins interact with a similar set of cellular proteins as E6 and E7 proteins encoded by HPVs that have been linked to human carcinogenesis and/or have transforming activities in vitro.

Molecular archeological evidence in support of the repeated loss of a papillomavirus gene.

It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.

Identification of a novel human papillomavirus by metagenomic analysis of vaginal swab samples from pregnant women.

BACKGROUND: The number of members in the genus Gammapapillomavirus of Family Papillomaviridae has recently been expanding most rapidly. The aim of this study was to characterize a novel human gammapapillomavirus type identified in a vaginal swab from a 25-year-old pregnant woman suffering from vaginitis. METHODS: Viral metagenomics method was used to detect the viral sequences in 88 vaginal swab samples collected from 88 pregnant women with vaginitis. A novel papillomavirus, named HPV-ZJ01 (GenBank no. KX082661), was detected in one sample and its complete genome sequence was amplified by PCR and sequenced by Sanger walking. Phylogenetic analyses based on the complete genome and the L1 protein of HPV-ZJ01 and other representative human papillomaviruses were done, respectively. Further PCR screening was performed in vaginal swabs (n = 135), cervical smears (n = 40) and cervical cancer tissues (n = 40) using nested-PCR primers designed based on HPV-ZJ01 sequence to investigate the prevalence of HPV-ZJ01. RESULTS: The genome of HPV-ZJ01 is 7,358 bp in length with a GC content of 37.8 %. HPV-ZJ01 was predicted to contain six open reading frames (E6, E7, E1, E2, L2, and L1) and a non-coding long control region (LCR). The genome shared the highest overall similarity to HPV-166, with 70.6 % nucleotide sequence identity while its L1 gene shared the highest nucleotide similarity to HPV-162, with 71.1 % sequence identity. Phylogenetic analysis suggested that HPV-ZJ01 belongs to a novel HPV type in the Gamma-PV genus, species Gamma-19, already containing HPV161, HPV162 and HPV166. PCR screening results indicated that none of the other samples were positive for HPV-ZJ01 except the original HPV-ZJ01 positive vaginal swab specimen. CONCLUSION: The genome sequence of a novel type of species Gamma-19 HPV was characterized. The screening PCR results suggested that HPV-ZJ01 is not associated with any of the cervical cancer samples tested. In order to confirm the prevalence and disease association, if any, for HPV-ZJ01, a further study with different sample types and a larger sample size is needed.

Characterization of novel human papillomavirus types 157, 158 and 205 from healthy skin and recombination analysis in genus γ-Papillomavirus.

Gammapapillomavirus (γ-PV) is a diverse and rapidly expanding genus, currently consisting of 79 fully characterized human PV (HPV) types. In this study, three novel types, HPV157, HPV158 and HPV205, obtained from healthy sun-exposed skin of two immunocompetent individuals, were amplified by the "Hanging droplet" long PCR technique, cloned, sequenced and characterized. HPV157, HPV158 and HPV205 genomes comprise 7154-bp, 7192-bp and 7298-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2). Phylogenetic analysis of the L1 ORF placed all novel types within the γ-PV genus: HPV157 was classified as a new member of species γ-12 while HPV158 and HPV205 belong to species γ-1. We then explored potential recombination events in genus γ-PV with the RDP4 program in a dataset of 74 viruses (71 HPV types with available full-length genomes and the 3 novel types). Two events, both located in the E1 ORF, met the inclusion criterion (p-values <0.05 with at least four methods) and persisted in different ORF combinations: an inter-species recombination in species γ-8 (major and minor parents: species γ-24 and γ-11, respectively), and an intra-species recombination in species γ-7 (recombinant strain: HPV170; major and minor parents: HPV-109 and HPV-149, respectively). These findings were confirmed by phylogenetic tree incongruence analysis. An additional incongruence was found in members of species γ-9 but it was not detected by the RDP4. This report expands our knowledge of the family Papillomaviridae and provides for the first time in silico evidence of recombination in genus γ-PV.

Complete genome sequence of a novel human papillomavirus identified by metagenomic analysis from a child with diarrhea in China.

A highly divergent human papillomavirus, named HPV-CH2, was identified in fecal samples from children with diarrhea in China by 454 high-throughput sequencing. Here, we report the complete genome sequence and genetic organization of the virus. The full-length nucleotide sequence of HPV-CH2 shares the highest sequence similarity with HPV-156, with 72 % nucleotide sequence identity. The L1 gene of the HPV-CH2 shared <70 % nucleotide identity with previously reported HPVs, suggesting HPV-CH2 as a new type of papillomavirus. Phylogenetic analysis revealed that the HPV-CH2 belongs to the genus Gammapapillomavirus. No HPV-CH2 was detected by PCR in samples from children with both gastroenteritis and respiratory infection.

Human papillomavirus-associated diseases.

The human papillomavirus (HPV) may be associated with various oral, genital, and cutaneous conditions, both benign and malignant. The association between sexually transmitted α-HPV types is the strongest with cervical cancer because almost all such malignancies contain viral DNA, notably HPV types 16 and 18. The contribution of cancer causing HPV types in other anogenital, oral, and oropharyngeal malignancies, plus benign disorders, is lower and with a less significant public health concern. Cervical cytologic screening is a well-established preventive measure that allows early detection and successful treatment of precancerous cervical lesions. In cases of all other HPV-associated disorders, early detection of a precancerous lesion is either difficult or almost impossible. HPV vaccination remains the only preventive measure against most HPV-related diseases.

Characterization of human papillomavirus type 154 and tissue tropism of gammapapillomaviruses.

The novel human papillomavirus type 154 (HPV154) was characterized from a wart on the crena ani of a three-year-old boy. It was previously designated as the putative HPV type FADI3 by sequencing of a subgenomic FAP amplicon. We obtained the complete genome by combined methods including rolling circle amplification (RCA), genome walking through an adapted method for detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR), long-range PCR, and finally by cloning of four overlapping amplicons. Phylogenetically, the HPV154 genome clustered together with members of the proposed species Gammapapillomavirus 11, and demonstrated the highest identity in L1 to HPV136 (68.6%). The HPV154 was detected in 3% (2/62) of forehead skin swabs from healthy children. In addition, the different detection sites of 62 gammapapillomaviruses were summarized in order to analyze their tissue tropism. Several of these HPV types have been detected from multiple sources such as skin, oral, nasal, and genital sites, suggesting that the gammapapillomaviruses are generalists with a broader tissue tropism than previously appreciated. The study expands current knowledge concerning genetic diversity and tropism among HPV types in the rapidly growing gammapapillomavirus genus.

Analysis of the genetic diversity and phylogenetic relationships of putative human papillomavirus types.

More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.

Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.

Nine complete genome sequences of cutaneous human papillomavirus genotypes isolated from healthy skin of individuals living in rural He Nan province, China.

Nine novel human papillomavirus (HPV) types were isolated from healthy skin of individuals in rural Anyang, China. All of these isolates belong to the genus Gammapapillomavirus. These data will provide us with useful information for a better understanding of PV evolution and the relationship of PV with the host.

One virus, one lesion--individual components of CIN lesions contain a specific HPV type.

In 20-40% of cervical intra-epithelial neoplasia (CIN) and in 4-8% of cervical carcinoma tissue specimens, multiple HPV genotypes have been detected. Whole tissue section (WTS) PCR does not determine how the individual types relate causally to complex and multiple CIN. Our objective was to determine whether laser capture micro-dissection (LCM) with HPV PCR genotyping (LCM-PCR) could accurately recover type-specific HPV DNA from epithelial cells in individual areas of CIN and normal epithelium, and whether one or more viruses are present in one lesion. For that, histologically selected samples of CIN and normal epithelium were isolated by LCM and analysed by the SPF(10) PCR/LiPA(25) (version 1) HPV genotyping system for 25 HPV genotypes. HPV genotypes detected in 756 areas of CIN (grade 1, 2 or 3) by LCM-PCR were compared with results obtained by WTS-PCR in 60 cases (74 biopsies). We showed that when a single HPV type is detected by WTS-PCR, that type was almost always (94%; 29/31) recovered by LCM-PCR from CIN. When multiple HPV types were present by WTS-PCR, their distribution within histological sections could be mapped by LCM-PCR. Association of a single HPV type with a discrete area of CIN was found for 93% (372/399) of LCM fragments analysed by PCR. We found colliding CIN lesions associated with separate HPV types and only 62% (61/99) of HPV types detected by WTS-PCR were found in CIN by LCM-PCR. Therefore, the LCM-PCR technique was found very accurate for high-resolution HPV genotyping and for assigning an individual HPV type to an area of CIN. At LCM level, in cervical biopsy sections with multiple HPV infections, the relation between HPV types and CIN lesions is often complex. Almost every HPV type found in CIN by LCM-PCR is associated with a biological separate independent CIN lesion-one virus, one lesion.

Identification of a novel human gammapapillomavirus species.

By using random PCR amplification, shotgun sequencing and sequence similarity searches, we analysed nucleic acids present in cell cultures inoculated with samples from unexplained cases of encephalitis. We identified a divergent human papillomavirus (HPV) sequence originating from a rectal swab. The full genome was amplified by inverse PCR and sequenced. The prototype of the sixth gammapapillomavirus species, HPV116, was not found in the patient's cerebrospinal fluid or respiratory secretions, nor in culture supernatants from other unexplained cases of encephalitis, indicating that its identification in an encephalitis patient was accidental.

Genetic diversity of cutaneous human papillomaviruses.

Human papillomaviruses (HPVs) of the genera Betapapillomavirus and Gammapapillomavirus are common on human skin. Sequencing of subgenomic amplicons of cutaneous HPVs has revealed a large number of novel putative HPV types within these genera. Phylogenetic analysis based on these amplicons revealed 133 putative HPV types with <90 % sequence identity to any known HPV type or to each other. As there are already 34 characterized HPV types described within the genera Betapapillomavirus and Gammapapillomavirus, they appear to be the most genetically diverse of the HPVs, apparently comprising at least 167 different HPV types.

[Study on the genotyping of human papillomavirus using a new DNA liquid chip in women of high-risk group of Shandong province].

OBJECTIVE: To evaluate the diagnostic applicability of human papillomavirus (HPV) liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong province. METHODS: To detect HPV genotypes on a 96-well plate with the liquid chip which can simultaneously detect and identify 26 common HPV genotypes in a total of 2925 cervical scrapes obtained from gynecological outpatients as well as to analyze the relationship between HPV types and different cervical diseases by studying the distribution of HPV genotypes and pathologic diagnosis. RESULTS: Among 639 cases who performed pathologic/cytological and histological diagnoses, 184 cases are in group of normal cytology, 266 cases in group of, 77 cases in group of cervical intra-epithelial neoplasia (CIN) I, 7 cases in group of CIN I - II, 46 cases in group of CIN I - II, 46 cases in group of CIN I - II and 13 cases in group of cervical cancer. The overall incidence of HPV in our samples is 36.0% (1054/2925) and 23 types of all 26 types on liquid chip are found. The most common genotypes found are HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18 (8.87%) and HPV-11 (6.94%). Among all the positive types, 87.32% are high-risk HPV and 13.68% are low-risk HPV genotypes. Both single and multiple types are easily identified, showing 66.22% ( n = 698) single type and 33.78% ( n = 356) multiple types. Of all the 1054 HPV-positive cases, 261 (24.8%) is occupied by women 21 to 25 years of age and progressively lower by older age groups, reaching 4.9% by women between 51 to 67 years old. The incidence of HPV in our samples is 23.37%, 33.08%, 54.54%, 57.14%, 82.61%, 91.30% and 100% for normal cytology, inflammation,CIN I ,CIN I - II, CIN II ,CIN III, and carcinomas specimens, respectively. Infections with more that one virus are common, accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I - II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II, III are 88.57%, 76.63%, 68.89% and 92.16% respectively. Conclusion The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. The HPV-positive rate increased along with the increase of grading on cervical lesions. There are more younger women among all the HPV-positive ones. Multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.