Genomic organization of a Gamma-6 papillomavirus metagenomic discovered from vaginal swab samples of Chinese pregnant women.

A complete genome sequence of human papillomaviruses (HPV) named as HPV-ujs-21015 was determined by viral metagenomic and PCR methods. The complete genome is 7354 bp in length with GC content of 41.7%, of which the genome was predicted to contain six ORFs (Open Reading Frame, ORF) coding for four early proteins (E7, E1, E4, and E2) and two late proteins (L1 and L2). Phylogenetic analysis based on the complete genome and the L1 protein showed that HPV-ujs-21015 belongs to a type 214 member within genus Gamma-6 papillomavirus. It is the first complete genome of Gamma-6 papillomavirus discovered from pregnant women in China.

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.

Cross-sectional associations between cutaneous viral infections and regulatory T lymphocytes in circulation.

BACKGROUND: Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood. OBJECTIVES: To examine cross-sectional associations between cutaneous viral infections and circulating forkhead-box P3 (FOXP3)-expressing T-regulatory (Treg) cells, suppressive cells that dampen effective antitumour immunity. MATERIALS AND METHODS: Blood, eyebrow hair (EBH) and skin swab (SSW) samples were collected from 352 patients 60 years and older undergoing skin screening, without prevalent skin cancer, while participating in an ongoing prospective cohort study of cutaneous viral infections and skin cancer. DNA corresponding to 98 cutaneous human papillomavirus (HPV) types and five human polyomaviruses (HPyV) was assessed in EBH and SSW. Distinct classes of circulating Treg-cell subpopulations were defined by flow cytometry including cutaneous lymphocyte antigen (CLA) and CCR4high Treg cells, both previously associated with cutaneous diseases. Age- and sex-adjusted associations between circulating T-cell populations and infection were estimated using logistic regression. RESULTS: Total Treg-cell proportion in peripheral blood was not associated with ß HPV or HPyV infection. However, the proportion of circulating CLA+ Treg cells was inversely associated with γ HPV EBH infection [odds ratio (OR) 0·54, 95% confidence interval (CI) 0·35-0·84]. Interestingly, circulating Treg cells expressing markers indicative of antigen activation (CD27- CD45RA- FOXP3+ CD4+ ) were also inversely associated with γ HPV infection in SSW (OR 0·55, 95% CI 0·30-0·99) and EBH (OR 0·56, 95% CI 0·36-0·86). CONCLUSIONS: Inverse associations between circulating Treg cells and γ HPV infection suggest that localized viral infection may promote immunosuppressive cell migration into skin.

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.

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.

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

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

Characterization of two novel gammapapillomaviruses, HPV179 and HPV184, isolated from common warts of a renal-transplant recipient.

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

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.

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.

Characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals.

BACKGROUND: Despite the strong evidence of HPV infection as the etiological agent in a subset of oral cancer, oral α-HPV detection is rare in healthy individuals, and little is known of the existing of novel HPV types in oral cavity. OBJECTIVE: We determined whether novel HPV types can be isolated from oral rinse samples collected from healthy individuals. STUDY DESIGN: We performed rolling circle amplification (RCA) coupled with degenerated PCR assay on 48 oral rinse samples to amplify novel HPV types. Full length HPV DNA was cloned using long range PCR. Quantitative type specific Taqman assays were used to determine the prevalence of novel HPV types in 158 archived oral tissue samples. RESULTS: We were able to isolate four novel human papillomavirus types. Full length HPV DNA was cloned for three of the four novel HPV types. All four HPV types belong to the genus Gammapapillomavirus (γ-PV), where HPV 171 is most closely related to HPV 169, showing 88% similarity; HPV 172 is most closely related to HPV 156, showing 70% similarity; HPV 173 is most closely related to HPV 4, showing 73% similarity; oral sample lavage (OSL) 37 is most closely related to HPV 144, showing 69% similarity. Finally, we showed that HPV 173 was rarely present in oral tissues (2/158), HPV 172 was only detected in normal oral tissues (25/76), and HPV 171 was more prevalent in malignant oral tissues (17/82 vs. 10/76, p=0.21). CONCLUSIONS: Novel γ-HPV types are present in oral cavity of healthy individuals.

Oral human papillomavirus detection in older adults who have human immunodeficiency virus infection.

OBJECTIVE: To evaluate reproducibility of oral rinse self-collection for human papillomavirus (HPV) detection and investigate associations between oral HPV, oral lesions, immune and sociodemographic factors, we performed a cross-sectional study of older adults with human immunodeficiency virus (HIV) infection. STUDY DESIGN: We collected oral rinse samples from 52 subjects at 2 different times of day, followed by an oral examination and interview. We identified HPV with the use of polymerase chain reaction platforms optimized for detection of mucosal and cutaneous types. RESULTS: Eighty-seven percent of individuals had oral HPV, of which 23% had oncogenic alpha, 40% had nononcogenic alpha, and 46% had beta or gamma HPV. Paired oral specimens were concordant in all parameters tested. Significant associations observed for oral HPV with increased HIV viral load, hepatitis C seropositivity, history of sexually transmitted diseases, and lifetime number of sexual partners. CONCLUSIONS: Oral cavity may be a reservoir of subclinical HPV in older adults who have HIV infection. Understanding natural history, transmission, and potential implications of oral HPV warrants further investigations.

Case-control study of cutaneous human papillomavirus infection in Basal cell carcinoma of the skin.

Genus-ß human papillomavirus (HPV) DNA has been detected in basal cell carcinoma (BCC) tumors, but most epidemiologic studies have not observed associations between genus-ß HPV seropositivity and BCC. A clinic-based case-control study was conducted to investigate cutaneous HPV infection in BCC. BCC cases (n=224) were recruited from a dermatology clinic, and controls (n=300) were patients who were screened negative for skin cancer. Antibodies against cutaneous HPV types in genera α, ß, γ, mu, and nu were measured, and tumors from a subset of BCC cases (n=195) were tested for HPV DNA. Overall associations were observed between BCC and seropositivity for HPV types in genus-α (odds ratio (OR)=1.61; 95% confidence interval (CI)=1.11-2.35), γ (OR=1.78; 95% CI=1.22-2.60), and mu (OR=1.56; 95% CI=1.06-2.30). BCC cases with ß-HPV DNA in their tumors were more likely to be ß-HPV seropositive than controls (OR=1.76; 95% CI=1.03-3.01), with type-specific associations observed for HPV8 and HPV23, whereas no association was observed between ß-HPV seropositivity and ß-HPV DNA-negative BCC. No concordance between seropositivity and tumor DNA status was observed for HPV types in genera α and γ. In conclusion, the combined serology and tumor DNA results suggest that ß HPV types may have a role in BCC. Additional studies of BCC that assess HPV types in multiple genera are needed.

B7-H1 expression model for immune evasion in human papillomavirus-related oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) is associated with oropharyngeal squamous cell carcinomas. Persistent viral infection is postulated to lead to carcinogenesis, although infection of benign adjacent epithelium is not typically observed. It is known that immune evasive tumor cells can provide an ideal niche for a virus. The B7-H1/PD-1 cosignaling pathway plays an important role in viral immune evasion by rendering CD8+ cytotoxic T cells anergic. We hypothesized that HPV-related oropharyngeal squamous cell carcinomas express B7-H1 as a mechanism for immune evasion. A tissue microarray was utilized, for which HPV E6/E7 mRNA by in situ hybridization was previously performed. Immunohistochemistry was performed to detect B7-H1 and staining was characterized by pattern, distribution, and intensity. B7-H1 was expressed by 84 of the 181 (46.4%) cases. Both tumor cell membranous and cytoplasmic expression were present and cytoplasmic expression was identified in some peritumoral lymphocytes. Expression was analyzed in several different ways and then considered binarily as positive versus negative. Tumors expressing B7-H1 were more likely to be HPV positive (49.2 vs. 34.1 %, p = 0.08). B7-H1 expression showed no correlation with disease recurrence in the entire cohort (OR = 1.09, p = 0.66), HPV positive cohort (OR = 0.80, p = 0.69) or HPV negative cohort (OR = 2.02, p = 0.22). However, B7-H1 expression intensity did correlate with the development of distant metastasis (p = 0.03), and B7-H1 intensity of 3+ (versus all other staining) showed a strong trend towards distant metastasis in the HPV positive (OR = 6.67, p = 0.13) and HPV negative (OR = 9.0, p = 0.13) cohorts. There was no correlation between B7-H1 expression and patient survival for any of the different ways in which staining was characterized, whether binarily, by distribution, intensity, or combined scores. B7-H1 is expressed in the majority of oropharyngeal squamous cell carcinomas with transcriptionally-active HPV. This suggests that B7-H1 expression by tumor cells may play a role in harboring persistent HPV infection.