Genome announcement : complete genome sequence of a novel Mupapillomavirus, HPV204

Human papillomaviruses (HPVs) are small, non-enveloped viruses with a circular double-stranded DNA genome, etiologically associated with various benign and malignant neoplasms of the skin and mucosa. As of May 30, 2015, 201 different HPV types had been completely sequenced and officially recognized and divided into five PV-genera: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. The Mupapillomavirus genus currently consists of only two HPV types: HPV1 and HPV63, identified in 1980 and 1993, respectively, both associated with sporadic cases of cutaneous warts. In this preliminary study, we announce the complete genome sequence of a novel HPV type, now officially recognized as HPV204. Based on preliminary data, the genome of HPV204 comprises a total of 7,227 bp and contains five early open reading frames (E1, E2, E4, E6, and E7) and two late ORFs (L1 and L2). No E5 ORF could be identified. Preliminary HPV204 clusters to the Mu-PV genus, species Mu-3.Fil: Kocjan, Boštjan J. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Šterbenc, Anja. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Hošnjak, Lea. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; SloveniaFil: Chouhy, Diego. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Bolatti, Elisa María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Giri, Adriana Angélica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área de Virología; ArgentinaFil: Poljak, Mario. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Sloveni

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a singletube ‘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.Fil: Chouhy, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Kocjan, Boštjan J.. University of Ljubljana; EsloveniaFil: Staheli, Jeannette P.. Seattle Children’s Research Institute; Estados UnidosFil: Bolatti, Elisa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hošnjak, Lea. University of Ljubljana; EsloveniaFil: Sagadin, Martin. University of Ljubljana; EsloveniaFil: Giri, Adriana Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rose, Timothy M.. Seattle Children’s Research Institute; Estados UnidosFil: Poljak, Mario. University of Ljubljana; Esloveni

Characterization of Novel Cutaneous Human Papillomavirus Genotypes HPV-150 and HPV-151

DNA from two novel HPV genotypes, HPV-150 and HPV-151, isolated from hair follicles of immuno-competent individuals, was fully cloned, sequenced and characterized. The complete genomes of HPV-150 and HPV-151 are 7,436-bp and 7,386-bp in length, respectively. Both contain genes for at least six proteins, namely E6, E7, E1, E2, L2, L1, as well as a non-coding upstream regulatory region located between the L1 and E6 genes: spanning 416-bp in HPV-150 (genomic positions 7,371 to 350) and 322-bp in HPV-151 (genomic positions 7,213 to 148). HPV-150 and HPV-151 are phylogenetically placed within the Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. As in other members of this genus, the intergenic E2-L2 region is very short and does not encode for an E5 gene. Both genotypes contain typical zinc binding domains in their E6 and E7 proteins, but HPV-151 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of the novel genotypes, quantitative type-specific real-time PCR assays were developed. The 95% detection limits of the HPV-150 and HPV-151 assays were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (total of 540 samples) revealed that HPV-150 and HPV-151 are relatively rare genotypes with a cutaneous tropism. Both genotypes were found in sporadic cases of common warts and SCC and BCC of the skin as single or multiple infections usually with low viral loads. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. A partial L1 sequence of a putative novel HPV genotype, related to HPV-150, was identified in a squamous cell carcinoma of the skin obtained from a 64-year old immuno-compromised male patient

Characterization of a Novel Cutaneous Human Papillomavirus Genotype HPV-125

The DNA genome of a novel HPV genotype, HPV-125, isolated from a hand wart of an immuno-competent 19-year old male was fully cloned, sequenced and characterized. The full genome of HPV-125 is 7,809-bp in length with a GC content of 46.4%. By comparing the nucleotide sequence of the complete L1 gene, HPV-125 is phylogenetically placed within cutaneotrophic species 2 of Alphapapillomaviruses, and is most closely related to HPV-3 and HPV-28. HPV-125 has a typical genomic organization of Alphapapillomaviruses and contains genes coding for five early proteins, E6, E7, E1, E2 and E4 and two late capsid proteins, L1 and L2. The genome contains two non-coding regions: the first located between the L1 and E6 genes (nucleotide positions 7,137–7,809, length 673-bp) and the second between genes E2 and L2 (nucleotide positions 3,757–4,216, length 460-bp). The E6 protein of HPV-125 contains two regular zinc-binding domains at amino acid positions 29 and 102, whereas the E7 protein exhibits one such domain at position 50. HPV-125 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of HPV-125, a quantitative type-specific real-time PCR was developed. The 95% limit-of-detection of the assay was 2.5 copies per reaction (range 1.7–5.7) and the intra- and inter-assay coefficients of variation were 0.47 and 2.00 for 100 copies per reaction, and 1.15 and 2.15 for 10 copies per reaction, respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (a total of 601 samples) showed that HPV-125 is a relatively rare HPV genotype, with cutaneous tropism etiologically linked with sporadic cases of common warts

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

Molecular methods for identification and characterization of novel papillomaviruses.

Papillomaviruses (PV) are a remarkably heterogeneous family of small DNA viruses that infect a wide variety of vertebrate species and are etiologically linked with the development of various neoplastic changes of the skin and mucosal epithelia. Based on nucleotide similarity, PVs are hierarchically classified into genera, species and types. Novel human PV (HPV) types are given a unique number only after the whole genome has been cloned and deposited with the International HPV Reference Center. As of March 09, 2015, 200 different HPV types, belonging to 49 species, had been recognized by the International HPV Reference Center. In addition, 131 animal PV types identified from 66 different animal species exist. Recent advances in molecular techniques have resulted in an explosive increase in the identification of novel HPV types and novel subgenomic HPV sequences in the last few years. Among PV genera, the Gamma-PV genus has been growing most rapidly in recent years with 80 completely sequenced HPV types, followed by Alpha- and Beta-PV genera that have 65 and 51 recognized HPV types, respectively. We reviewed in detail the contemporary molecular methods most often used for identification and characterization of novel PV types, including polymerase chain reaction, rolling circle amplification and next generation sequencing. Furthermore, we present a short overview of 12 and 10 novel HPV types recently identified in Sweden and Slovenia, respectively. Finally, an update on the International Human Papillomavirus Reference Center is provided

Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63.

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1

Percentage similarity between individual genes (E6, E7, E1, E2, E4, L1, L2) of HPV179 and HPV184 and phylogenetically related HPV types.

<p>Percentage similarity between individual genes (E6, E7, E1, E2, E4, L1, L2) of HPV179 and HPV184 and phylogenetically related HPV types.</p

DISTRIBUTION OF HUMAN PAPILLOMA VIRUS GENOTYPES IN WOMEN WITH CERVICAL CANCER IN SLOVENIA AND GENOMIC VARIANTS OF HPV 16, HPV 18 AND HPV 33

Background. To establish the distribution of human papillomavirus (HPV) genotypes in representative population of women with cervical cancer (CC) in Slovenia in order to contribute the lacking data on HPV in CC and to assess the potential local benefit of future prophylactic HPV vaccination. Furthermore, we wanted to determine genomic variants of the most common HPV genotypes. Methods. Polymerase chain reaction with GP5+/GP6+ primers was performed in all 278 CC samples for HPV DNA detection and genotyping. Negative samples were additionally tested using CPI/CPIIg primers and INNO-LiPA HPV genotyping assay. Genomic variants of HPV 16, HPV 18 and HPV 33 were determined by sequencing of LCR, E6 and E7 genetic regions. Results. A total of 262/278 CC samples (94.2 %) were HPV DNA positive. HPV genotypes in Slovenian women with CC, in decreasing order of frequency, were: 16, 18, 33, 45, 31, 51, 58, 59, 35, 52, 73 and 82. Detailed genomic analysis was carried out on 40/178 isolates of HPV 16, 20/34 isolates of HPV 18 and 11/13 isolates of HPV 33. A total of 26 genomic variants of HPV 16 were identified. Thirty-eight isolates (95 %) belonged to the European branch; one isolate (2.5 %) belonged to the Asian-American branch and one (2.5 %) to African branch.1, 2 A total of 18 genomic variants of HPV 18 were identified. Nineteen isolates (95 %) belonged to the European branch and one isolate (5 %) belonged to the African branch.2, 3 Seven genomic variants of HPV 33 were identified. Five isolates (45.5 %) belonged to prototypic variants and 6 (54.5 %) belonged to non-prototypic variants.4 Conclusions. Distribution of all HPV genotypes in Slovenian women with CC was in the present study established for the first time and represents baseline distribution before mandatory HPV vaccination. Prophylactic HPV vaccination with currently available vaccines could prevent up to 77.1 % of CC in Slovenia caused by HPV 16 or HPV 18. Almost all isolates of HPV 16 and HPV 18 belonged to European branches; prototypic and non-prototypic HPV 33 variants were almost equally distributed among Slovenian patients with CC